AU2004248859C1 - Antiviral acylguanidine compounds and methods - Google Patents

Abstract

The invention relates to compounds having antiviral activity and methods utilising the compounds to treat viral infections.

Description

WO 2004/112687 PCT/AU2004/000866 -1 ANTIVIRAL COMPOUNDS AND METHODS FIELD OF INVENTION The present invention relates to methods for retarding, reducing or otherwise 5 inhibiting viral growth and/or functional activity. The invention also relates to compounds and compositions suitable for use in the methods. BACKGROUND OF THE INVENTION Currently, there is a great need for the development of new treatments that are effective against viral infections, particularly against viral infections which are 10 associated with high morbidity and mortality, and which impact on sizable populations. Treatments currently available are inadequate or ineffective in large proportions of infected patients. For example, in ameliorating AIDS symptoms and prolonging life expectancy, a measure of success has been achieved with drugs targeting the viral reverse 15 transcriptase and protease enzymes (Miller and Sarver, 1997; Mitsuya, 1992; Moore, 1997; and Thomas and Brady, 1997). However, no single treatment method is completely effective against HIV infection, (Barry et al, 1998; Decks, 1998; Miles, 1997; Miles, 1998; Moyle et al, 1998; Rachlis and Zarowny, 1998; Veil et al, 1997; Volberding and Decks, 1998; and Volberdin, 1998). 20 PCT application PCT/AU99/00872 describes the use of compounds 5-(NN hexamethylene)-amilorlde and 5-(N,N-dimethyl)-aniloride in the treatment of HIV infection. Another virus considered to be a significant human pathogen is the Hepatitis C virus (HCV). This is a significant human pathogen in terms of both cost to human 25 health and associated economic costs. 1CV causes chronic hepatitis and cirrhosis and is the leading indicator for liver replacement surgery. In 2002 the Centre for Disease Control and Prevention estimated that more than 4 million people were infected in the USA alone and that approximately 8,000 to 10, 000 die as a result of chronic HCV infection yearly. There is no known cure or vaccine. More effective 30 pharmacological agents are urgently required.

WO 2004/112687 PCT/AU2004/000866 -2 A further well-known family of pathogenic viruses are the Coronaviruses. Coronaviruses (Order Nidovirales, family Coronaviridae, Genus Coronavirus) are enveloped positive-stranded RNA viruses that bud from the endoplasmic reticulum Golgi intermediate compartment or the ds-Golgi network (Fischer, Stegen et al. 5 1998; Maeda, Maeda et al. 1999; Corse and Machamer 2000; Maeda, Repass et al. 2001; Kuo and Masters 2003). Coronaviruses infect humans and animals and it is thought that there could be a coronavirus that infects every animal. The two human coronaviruses, 229E and OC43, are known to be the major causes of the common cold and can occasionally 10 cause pneumonia in older adults, neonates, or immunocompromised patients (Peiris, Lai et al. 2003). Animal coronaviruses cin cause respiratory, gastrointestinal, neurological, or hepatic diseases in their host (Peiris, Lai et al. 2003). Several animal coronavirus are significant vetornary pathogens (Rota, Oberste et al. 2003). Severe acute respiratory syndrome (SARS) is caused by a newly identified 15 virus. SARS is a respiratory illness that has recently been reported in Asia, North America, and Europe (Peiris, Lai et al. 2003). The causative agent of SARS was identified as a coronavims. (Drosten, Gunther et al. 2003; Ksiazek, Erdman et al. 2003; Peiris, Lai et al. 2003). The World Health Organization reports that the cumulative number of reported probable cases of SARS from 1 November 2002 to 20 the 1 ' July 2003 is 8,437 with 813 deaths, nearly a 10% death rate. It is believed that SARS wili not be eradicated, but will cause seasonal epidemics like thecold or influenza viruses (Vogel 2003). To improve the prospect of treating and preventing viral infections, there is an on-going need to identify molecules capable of inhibiting various aspects of the viral 25 life cycle. It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative. Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of 30 common general knowledge in the field.

-3 SUMMARY OF THE INVENTION The inventors have surprisingly found that certain compounds that fall under the classification of substituted acylguanidines have antiviral activity against viruses from a 5 range of different virus families. Without intending to be bound by any particular theory or mechanism of action, and despite current dogma, it appears possible that viral replication can be retarded by inhibiting or otherwise down-regulating the activity of ion channels expressed in the host cell. Thus, the negative impact of the compounds of the present invention on viral replication may be mediated by the inhibition or otherwise 10 down-regulation of a membrane ion channel relied upon by the virus for replication. This membrane ion channel may be a viral membrane ion channel (exogenous to the host cell) or a host cell ion channel induced as a result of viral infection (endogenous to the host cell). As an example, the compounds of the present invention may inhibit Vpu or p7 15 function and thereby inhibit the continuation of the respective HIV or HCV life cycle. The SARS virus encodes an E protein which is shown for the first time, by the present inventors, to act as an ion channel. As similar E proteins are present in other coronaviruses, the compounds, compositions and methods of the present invention would have utility in the inhibition and/or treatment of infections by other coronaviruses. 20 The present invention is concerned with novel antiviral compounds that fall under the classification of substituted acylguanidines. It does not include in its scope the use of compounds 5-(N,N-hexamethylene)amiloride and 5-(N,N-dimethyl)-amiloride for retarding, reducing or otherwise inhibiting viral growth and/or functional activity of HIV. 25 According to a first aspect, the present invention provides an antiviral compound of Formula I O N' R3 R, ' N ' N ,R H I or a pharmaceutically acceptable salt thereof, 30 wherein, -4 Ri= -R RL Rg RL R R Rk or ; R2, R3 and R 4 are independently hydrogen, O O P.O 0 -~0 N . ~0 (E) N -4a N NB N N N F F or F F and wherein X = hydrogen, hydroxy, nitro, Ci-6alkyl, C3.cycloalkyl, phenyl, C 1

-

6 alkeneyl, C3..cycloalkeneyl, Ci- 6 alkeneoxy, or benzo; RdK, Re, Rf, R , Rk , RL, Rm, R. , R., R, independently = hydrogen, amino, C1.

5 alkyl, hydroxy, aryl, substituted aryl, substituted amino, mono or dialkyl substituted amino, cycloalkyl-substituted amino, aryl-substituted amino, H3C

H

2 N N

NH

2 O or PrS; Rg , Ri independently = hydrogen, hydroxy or C 1

.

5 alkyl; Rj = hydrogen, amino, CI.

5 alkyl, hydroxy, aryl, substituted aryl, substituted amino, alkyl-substituted amino, cycloalkyl-substituted amino, aryl-substituted amino, PrS,

H

2 N N ' or NH 2 0 when R 1 is C 6

H

5 CH=CH, R2 is hydrogen and R 3 is phenyl, l4 cannot be phenyl; when R 1 is phenyl, R2 is hydrogen, and R3 is benzoyl, R 4 cannot be benzoyl; when R, is phenyl, R2 is substituted benzyl, R3 is hydrogen and R4 is hydrogen, R., Ro and R, cannot all be hydrogen; when RI is phenyl, R 3 is hydrogen and l4 is hydrogen, R 2 cannot be benzyl or phenyl; when Ri is phenyl, R2 is hydrogen, R 3 cannot be phenyl together with 1 4 as benzoyl; when R, is phenyl, R2 is hydrogen, R3 and R4 cannot both be benzyl; wherein R2, R 3 , 14, Rj and Rk cannot all be hydrogen; and -4b wherein the compound is not: O NH 0 NH S NH 2 N NH 2 Me O NH 0 NH N NH2 N NH 2 H H OMe OH o NH 0 N H N NH 2 Me N NH 2 H H

NO

2 O NH o NH Me N NH 2 e N NH 2 H H O NH 0 NH e N NH 2 I - H H H- 0 2 N O NH 0 NH N NH 2 r MNN NH 2 H H M e A-MeG A O NH 0 NH N ' NH- 2 N ' NH 2 H H

F

3 C A-or Me 2 N A According to a second aspect, the present invention provides a pharmaceutical composition comprising an antiviral compound according to the first aspect and optionally one or more pharmaceutical acceptable carriers or derivatives. According to a third aspect, the present invention provides a method for reducing, retarding or otherwise inhibiting growth and/or replication of a virus comprising -4c contacting a cell infected with said virus or exposed to said virus with a compound according to the first aspect or a phamaceutical composition according to the second aspect. According to a fourth aspect, the present invention provides a method for preventing the infection of a cell exposed to a virus comprising contacting said cell with a compound according to the first aspect or a pharmaceutical composition according to the second aspect. According to a fifth aspect, the present invention provides a method for the therapeutic or prophylactic treatment of a subject infected with or exposed to a virus, comprising the administration of a compound according to the first aspect or a pharmaceutical composition according to the second aspect to a subject in need of said treatment. According to a sixth aspect, the present invention provides a method of down regulating a membrane ion channel functional activity in a cell infected with a virus, comprising contacting said cell with a compound according to the first aspect or a pharmaceutical composition according to the second aspect. According to a seventh aspect, the present invention provides a method of reducing, retarding or otherwise inhibiting growth and/or replication of a virus that has infected a cell, said method comprising contacting said infected cell with a compound according to the first aspect or a pharmaceutical composition according to the second aspect, wherein said compound or composition down regulates functional activity of a membrane ion channel derived from said virus and expressed in said infected cell. According to an eighth aspect, the present invention provides a method of reducing, retarding or otherwise inhibiting growth and/or replication of a virus that has infected a cell in a mammal, said method comprising administering to said mammal a compound according to the first aspect or a pharmaceutical composition according to the second aspect, wherein said compound or composition down regulates functional activity of a membrane ion channel expressed in said infected cell. According to a ninth aspect, the present invention provides a method for the therapeutic or prophylactic treatment of a subject infected with or exposed to a virus comprising administering to said subject a compound according to the first aspect or a pharmaceutical composition according to the second aspect, wherein said compound or -4d composition down-regulates functional activity of a membrane ion channel derived from said virus. According to a tenth aspect, the present invention provides an antiviral compound selected from the group consisting of: N,N'-bis(l -napthoyl)guanidine, N-(cinnamoyl)-N'phenylguanidine, (3-phenylpropanoyl)guanidine, N,N'-bis-(cinnamoyl)-N"-phenylguanidine, N-(3-phenylpropanoyl)-N'-phenylguanidine, N,N'-bis(3phenylpropanoyl)-N"-phenylguanidine, N-(6-Hydroxy-2-napthoyl)-N'-phenylguanidine, N,N'-Bis(amidino)napthalene-2,6-dicarboxamide, N"-Cinnamoyl-N,N'-diphenylguanidine, N,N'-Bis(3-phenylpropanoyl)guanidine, benzyoylguanidine, N-benzoyl-N'-cinnamoylguanidine, (5-Phenyl-penta-2,4-dienoyl)guanidine and (Quinoline-2-carbonyl)guanidine, or a pharmaceutically acceptable salt thereof According to an eleventh aspect, the present invention provides a pharmaceutical composition comprising a compound according to the tenth aspect, and optionally one or more pharmaceutical acceptable carriers or derivatives. According to a twelfth aspect, the present invention provides use of a compound according to the first aspect or tenth aspect for the manufacture of a medicament for: reducing, retarding or otherwise inhibiting growth and/or replication of a virus; preventing the infection of a cell exposed to a virus; the therapeutic or prophylactic treatment of a subject infected with or exposed to a virus; down regulating a membrane ion channel functional activity in a cell infected with a virus; reducing, retarding or otherwise inhibiting growth and/or replication of a virus that has infected a cell; -4e reducing, retarding or otherwise inhibiting growth and/or replication of a virus that has infected a cell in a mammal; or the therapeutic or prophylactic treatment of a subject infected with or exposed to a virus. An embodiment of the present invention provides an acylguanidine with antiviral activity. According to an embodiment, the present invention provides an antiviral compound of Formula I O N', R3 R2 Ri ~N <N H I wherein RI-R 4 are independently aromatic groups, heteroaromatic groups, alkylaromatic groups, alkylheteroaromatic groups, alkenylaromatic groups, alkenylheteroaromatic groups, cycloalkylaromatic groups, cycloalkylheteroaromatic groups, aryloxyalkyl groups, heteroaryloxyalkyl groups, said groups are mono or polycyclic, and are optionally substituted with one or more substitutents independently selected from hydrogen, hydroxy, nitro, halo, amino, substituted amino, alkyl-substituted amino, cycloalkyl-substituted amino, aryl-substituted amino, CI 6 alkyl, Ci 6 alkyloxy,

C

3 .scycloalkyl, halo-substituted Ci 16 alkyl, halo-substituted C 1 .6alkyloxy, phenyl, CI-alkeneyl, C 3

-

6 cycloalkeneyl, Ci- 6 alkeneoxy, benzo, aryl,

H

2 N N \ substituted aryl, PrS, O ,or NH 2 0 According to an embodiment, the present invention provides an antiviral compound of Formula I O N'R3 N~<N2 N N H 1 -4f or pharmaceutically acceptable salts thereof, wherein, R, Rb N N N" R.

WO 2004/112687 PCT/AU2004/000866 -5 R, N ((E) RL aRn Rk N I' OD 5 or; 100 and where A A N 0 0 0 0 (E) BOD'PY'I ofI 10N 1and he re indeednlyrgn WO 2004/112687 PCT/AU2004/000866 -6 X= hydrogen, hydroxy, nitro, halo, CI-salkyl, C 14 alkyloxy, C3.4cycloalkyl, halo-substituted C 1

.

6 alkyl, halo-substituted C. 5 alkyloxy, phenyl, C 14 alkeneyl, C 3

.

6 cycloalkeneyl, C 1 -alkeneoxy, or benzo; 5 R , R , Rd a, Re , Rf , Ra , Rk , RL , R,, Rd , Ro , R, independently= hydrogen, amino, halo, C1..salkyl, Cu.

5 alkyloxy, hydroxy, aryl, substituted aryl, substituted amino, mono or dialkyl-substituted amino, cycloalkyl-substituted amino, aryl-substituted amino,

H

3 C.

H

2 N N

NH

2 0 or PrS; 10 R, RIindependently =hydrogen, hydroxy, halo, or C .s alkyl; R= hydrogen, amino, halo, CIalkyl, Cialkyloxy, hydroxy, aryl, substituted aryl, substituted amino, alkyl-substituted amino, cyoloalkyl-substituted amino, aryl-sobstituted amino, PrS,

H

2 N N \ O , or

NH

2 0 15 Preferably, the compounds of the invention include the following: 5-(N,N-hcxamethylene)aiiloride comprising the structure 0 NH 2 Cl N 2 N NH2 NN NH2 20 WO 2004/112687 PCT/AU2004/000866 -7 5-(NN-Dimethyl)amiloride hydrochloride comprising the stmeture o NH 2 HC N NH2

CH

3 5-(N-methyl-N-isobutyl)amiloride comprising the structure 0 NH 2 C N N NH 2 CN N NH 2 51 5-(N-ethyl-N-isopropyl)amiloride (herein referred to as EIPA), comprising the structure o NH2 N N11 NHh C1 N NH WO 2004/112687 PCT/AU2004/000866 N-(3,5-Diamino-6-chloro-pyrazine-2-carbony)-N'-phenyl-guanidine, comprising the structure o N~H C)N: NN H2N N NH 2 N-Benzyl-N'-(3,5-diamino-6-choro-pyrzine-2-carbony)-guauidine, comprising the 5 structure o NHT 2 C1>N NA H Ha2 N NH2 3-methoxy amiloride comprising the structure comprising the structure o NH 2 ClXN: N>

NH

2

H

2 N OCH 3 3-methoxy-5-(N,N-Hexamethylene)-amiloride comprising the structure 0 NH2 Cl NA N NH 2 N ON

CU

3 10 WO 2004/112687 PCT/AU2004/000866 -9 3-hydroxy-5-hexamethyleneimino-amiloride comprising the stmotUre o NH2 C1 NNA N OH Hexamethyleneimino-6-phenyl-2-pyraxinecarboxamide comprising the structure 0 NH 2 NA N N NH2 N N NH 2 5 N-amidino-3,5-diamino-6-phenyl-2-pyrazinecarboxamide comprising the structure 0 NH 2 N ~N A NHN

NH

2 1 2 N N NH 2 10 WO 2004/112687 PCT/AU2004/000866 -10 5-(NN-hexamethylene)amiloride comprising the structure o NH 2 C N N NH 2 N NH 2 N-amidino-3-amino-5-phenyl-6-chloro-2-pyrazinecarboxamide comprising the 5 stroture o NH 2 Cl N Ar N N -kNH 2 W' NH2 394 DichloroBenzamil comprising the stmuture o

NH

2 Cl Nll 5NNE - Cl

NIT

2 N NIl 2 Ci 10 WO 2004/112687 PCT/AU2004/000866 -11 2'4 Dichlorofenzamil HCi comprising the structure 0 NH 2 C1 C1 Nl NH NH2 NH 2 Cl 5-(N-methyl-N-guanidinocarbonyl-methyl)amiloride comprising the structure O NH2 Cl N97 H3CN N NH2 N N Nit H2N N NH20 5 5-(N,N-Diethyl)amiloride hydrochloride comprising the structure N NH2 H13C % N NH2 H3C 5-(NN-Dimethyl)amiloride hydrochloride comprising the structure 0) NH.t C H3 A 2

HC

3 WO 2004/112687 PCT/AU2004/000866 -12 5-tert-butylamino-amiloride comprising the structure 0 NH 2 HNH 6-Iodoamiloride comprising the structure o NH 2

N

t

NH

2 5 H 2 N N

NH

2 Bodipy-FL Amiiloride comprising the structure o NH 2 C1 N N N'O H

H

2 N N NH 2 5-(4-fluorophenyl)amiloride comprising the structure 0 NH 2 F N N

NH

2 H 10 WO 2004/112687 PCT/AU2004/000866 -13 1-napthoylguanidine comprising the structure

H

2 N NH 2 0 N 2-napthoylguanidine comprising the structure 0 NH2

N>NH

2 11 N-(2-napthoyl)-N'-phenylguanidine comprising the structure o HN N N NNH N,N'-bis(2-napthoyl)guanidine comprising the structure o NH 2 O H H 10 WO 2004/112687 PCT/AU2004/000866 N,N'-bis(1-napthoyl)guanidine comprising the structure 0 NH20 N N H H NN'-bis(2-napthoyl)-N"-phenylguanidine comprising the structure 0 N N N N)< NH H 5 6-methoxy-2-naphthoylguanidine comprising the structure N NH 2 H N-Cinnamoyl-N',N-dimethylguanidine comprising the stmuture o NH HNNCH3 WO 2004/112687 PCT/AU2004/000866 -15 3-quinolinoylguatidine comprising the structure O NH2 N NH 2 N oinnmoylguanMdne comprising the structure o NH2 Nl

NH

2 5 4-phenylbenzoylguanidine comprising the structure 0 NH N N2 H N-(cinnamoyl)-N'phenylguanidine comprising the structure 0 NH N NH H 10 N WO 2004/112687 PCT/AU2004/000866 -16 (3-phenylpropanoy)guanidine comprising the structure oC NH

NA)NH

2 N,N'-bis-(cinnamoy1)-N"-phenylguanidine comnprisihg the stmucture O N N 5 N-(3-phnylpropanoyl)-N'-phenylguaidine comprising the stmeture AO NH H H N -bis(3phenylpr panoyl)-N"-phenylguanidine comprisin g the structure O N 0 H N H HR WO 2004/112687 PCT/AU2004/000866 -17 trans-3-furanacryoylguanidine comprising the structure 0 NH N NE2~ 0 2 N-(6-Hydroxy-2-napthoyl)-N-phenylguanidine comprising the structure ON NH H 5 - 5 (4-Phenoxybenzoyl)guanidine comprising the structure o NH2 10- NN-Bis(amnidino)napthalene-2,6-dicarboxamide comprising the structure O -NH2 N NH2 H2N N NH2 O 15 WO 2004/112687 PCT/AU2004/000866 1-Is 6-bromo-2-napthoylguanidine comprising the structure O NH N N NH 2 H Br 1-bromo-2-napthoylguanidine comprising the structure Br 0 NH N NH2 H 2-(2-naptbyl)acetoylguanidine comprising the structure H .,,N N 2 10 N"-Cinnamoyl-N,N'-diphenylguanidine comprising the structure O0 HNJ M N NH NH 15

NH

2 H 15 WO 2004/112687 PCT/AU2004/000866 -19 N,N'-Bis(3-phenylpropanoyl)guanidine comprising the structure o NH O N H Benzoylguanidine comprising the structure 0. NH N NH2 I H (4-Chlorophenoxy-acetyl]guanidine comprising the structure O NH A N NH 2 H C1~ 10 N-Benzoyl-N'-oinnamoylguanidine comprising the structure o NH 2 O ( N N [(E)-3-(4-Dimethylaminophenyl)-2-methylacryloylguanidine comprising the 15 structure 0 NH A N N NI 2 H N I WO 2004/112687 PCT/AU2004/000866 -20 (4-Cblorocinnamoyl)guanidine comprising the struture o NH2 -~ N NH 2 Cl (4-Bromocinnamoyl)guanidine comprising the structure o NH1 2 A N NH 2 Br (4-Methoxycinnamoyl)guanidine comprising the structure O N NH 2 N N (5-Pheypenta-2,4-dienoyl)guanidine comprising the structure e

A

2

,NH

2 10 W (3-Bromocinnamoyl)guanidine comprising the structure O NH Br" N NH2 H 1$ (3-Methoxycimnamoyl)guanidine comprising the structure o NH 0 N NH2

H

WO 2004/112687 PCT/AU2004/000866 -21 (3-Chlorocinnamoyl)guanidine comprising the structure 0 NH N NH 2 I H (2-Chlorocinnamoyl)guanidine comprising the structure O NH N KI 2 H C1 5 (2-Bromocinnamoyl)guanidine comprising the structure o NH N NH 2 H Br (2-Methoxycinnamoyl)guanidine comprising the structure o NH N NH 2 H 0 10 (trans-2-Phenylcyelopropanecarbonyl)guanidine comprising the structure o NH N NH2 H [3-(3-Pyridyl)acryloy]guanidine comprising the structure o NH . N NH 2 I H N 15 WO 2004/112687 PCT/AU2004/000866 .22 (4-Hydroxycinnamoyl)guanidine comprising the structure O NH (E) N NH2 H (Quinoline-2-carbonyl)guanidine comprising the structure o NH2 N N H2 (4-Nitrocinnamoyl)guanidine comprising the structure O NH N NH 2 H 0 2 N 10 (3-Nitrocinnamoyl)guanidine comprising the structure o NH N NH2 H H

NO

2 (2-Nitrocinnamoyl)guanidine comprising the structure o NH N

NH

2 1 H H

NO

2 15 WO 2004/112687 PCT/AU2004/000866 -23 (a-Methyloinnamoyl)guanidine comprising the structure o NH N N NH 2 H

CH

3 trans-3-(1-napthyl)aoryloylguanidine comprising the structure O NH N-

N

H

2 H 4-phenyleinnamoylguanidine comprising the structure o NH N NNH H H 10

CF

3 3-methyloinnamoylguanidine comprising the strccttre o NH N NH2 H

CH

3 WO 2004/112687 PCT/AU2004/000866 -24 4-(trifluoromethyl)cinnaoylguanidine comprising the structure O NH ' N N12 H H F3C 2-metbyleinnamoylguanidine comprising the structure

CH

3 0 NH N NH 2 H 5 2-(trifluoromethyl)cinnamoylguanidine comprising the structure

CF

3 0 NH N NH2 H H 4-methyleinnamoylguanidine comprising the structure O NH N NH 2 H H 10 H3C 4-isopropylcinnamoylguanidine comprising the structure O NH N NH2 H H3C H 15 WO 2004/112687 PCT/AU2004/000866 -25 3-fluorocimamoylguanidine composing the structure O NE N NH2 IH H 2-fluorocinnamoylguanidine comprising the structure o NH N NH 2 H F 4-fluorocinnamoylguanidine comprising the structure o NH 10 3,4-dichlorocinnamoylguanidine comprising the structure N NH 2 NH H H H C' 2,4-dichlorocinnamolyguanidine comprising the structure o NH N NH 2 H H 15 WO 2004/112687 PCT/AU2004/000866 -26 2,6-dichlorocimaamoylguanidine comprising the structure Cl 0 NH N NH 2 H -H Cl 4-ethoxycinnamoylguanidine comprising the stnoture 0 NH N N NH2 I H 5 H 3

CH

2 CO 3,4-(methylenedioxy)cinnanoylguanidine comprising the structure O NR N

NH

2 H 0 O 10- 3-(2-napthyl)acryloylguanidine comprising the structure 0 NH N NH2 4-t-butylcinnamoylguanidine comprising the structure 0 NH N NHI 2 H H3C.C H

H

3 C I CH3 15 WO 2004/112687 PCT/AU2004/000866 -27 3,4,5-trimethoxycinnamoylguanidine comprising the structure 0 NH

H

3 CO N NH2 H

OCH

3 2-(1-napthyl)acetoylguanidine comprising the structure O NH N

NH

2 H 5 2,5-dimethyleinnamoylguanidine comprising the structure 0 NH

H

3 C H H

CH

3 2,34ifluorocinnamoylguanidine comprising the structure o NH 'N NH 2 I H H F F 3-phenykcinnamoylguanidine comprising the strutuxe o NH

N

1 NH2 H -H 10 WO 2004/112687 PCT/AU2004/000866 -28 3-(trans-hept-1 -en-1-yl)cinnaMoylguanidine comprising the structure o NH N NH 2 H H 2-ethylcinnamoyguanidine comprising the structure o NI N

NH

2 H CHH 5 33 2-chloro-6-fluorocinnamoylguanidine comprising the structure F 0 NH NIZZ N N NH2 H H 10 3-t-butylcinnamoylguanidine comprising the structure O NE N NH2 H

H

3 C CH 3

CH

3 WO 2004/112687 PCT/AU2004/000866 -29 3,4-difluorocinnamoylguanidine comprising the structure O NH 'N NH2 H H H F 5-brono-2-fluorocinnamoylguanidine comprising the structure 0 NH Br N

NH

2 H 5 FH 3-(trifluoromethoxy)cinnamoylguanidine comprising the structure o NH NZ!N N NH 2 IH H

OCF

3 2-ethoxycinnamoylguanidine comprising the structure o NH N N N NH 2 H H H 10 001 2

CH

3 2-t-butylcinnamoylguanidine comprising the structure

H

3 C~ H3 2N Ut 0 NHl N : N NH2 H H 15 WO 2004/112687 PCT/AU2004/000866 -30 3-(cyclohex-1-en-1-yl)cinnamoylguanidine comprising the structue o NH N

NH

2 H H cinnamoylguanidine hydrochloride comprising the structure o NI N 2 4 H .HC1 5 2,3,5,6,-tetramethylhmamoylguanidine comprising the structure (Bit 134)

CH

3 0 NH

H

3 C N N N NH2 H H

CH

3

CH

3 2-cyclohexylcinnanoylguanidine comprising the structure o NH N N N NH 2 H 10 5-bromo-2-methoxycinnamoylguanidine comprising the structure 0 NH Br r NZ N4 N. NH9 2 H H OCH3 WO 2004/112687 PCT/AU2004/000866 -31 2,3-dimethy1cinnamoylguanidine comprising the structure o NH N z N N NH2 H

CH

3

CH

3 3-ethoxycinnamoylguanidine comprising the structure o NH. N NH2 I H H 5 OCH2CH 3 3-isopropylcinnamoylguanidine hydrochloride comprising the structure o NH N N NH2 H H .HCl 2-phenylcihnamoylguanidine comprising the structure o NH I H H 10 2-(cyclohex-1-en-lyl)cinnamoylguanidine comprising the structure o NH N NH 2 H H

H

WO 2004/112687 PCT/AU2004/000866 -32 2,4,6-trimethylcinnamoylguanidine comprising the structure

CH

3 0 NH N NH 2 H H H3C CH3 (5-Phenyl-penta-2,4-dienoyl)guanidine comprising the structure 0 NH N NH 2 H 5 - 5-(3'-bromophenyl)penta-2,4-dienoylguanidine comprising the structure O NH

NH

2 Br 10 5-(2' bromophenyl)penta-2,4-dienoylguanidine comprising the stmucture O NE N.H2 Furanacryloyl comprising the structure 0 NHl 0 N' NH 2 H 15 -33 Preferably, the compounds of the invention are capable of reducing, retarding or otherwise inhibiting viral growth and/or replication. Preferably, the antiviral activity of the compounds of the invention is against viruses such as those belonging to the Lentivirus family, and the Coronovirus family family of 5 viruses. For example, the compounds of the invention exhibit antiviral activity against viruses such as Human Immunodeficiency Virus (HIV), Severe Acute Respiratory Syndrome virus (SARS), Mouse Hepatitis virus (), and Hepatitis C virus (HCV). According to an embodiment of the present invention, there is provided a pharmaceutical composition comprising an antiviral compound according to the 10 embodiments, and optionally one or more pharmaceutical acceptable carriers or derivatives, wherein said compound is capable of reducing, retarding or otherwise inhibiting viral growth and/or replication. Preferably, the antiviral activity of the compounds of the invention is against viruses such as those belonging to the Lentivirus family, and the Coronovirus family of viruses. 15 For example, the compounds of the invention exhibit antiviral activity against viruses such as Human Immunodeficiency Virus (HIV), Severe Acute Respiratory Syndrome virus (SARS), Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus (BCV), Porcine Respiratory Coronavirus (PRCV), Hepatitis C virus (HCV) and Equine Arteritis Virus (EAV). 20 Other Coronaviruses which can be inhibited or their infections treated by the compounds of the invention are those listed in Table 1. The compositions of the invention may further comprise one or more known antiviral compounds or molecules. According to an embodiment, there is provided a method for reducing, retarding or 25 otherwise inhibiting growth and/or replication of a virus comprising contacting a cell infected with said Virus or exposed to said virus with a compound according to the embodiments. Preferably, the virus is from the Lentivirus family, or the Coronavirus family. More preferably, the virus is Human Immunodeficiency Virus (HIV), Severe Respiratory 30 Syndrome virus (SARS), Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus (BCV), Porcine Respiratory Coronavirus (PRCV), Mouse Hepatitis virus (MHV), Hepatitis C virus (HCV), or Equine Arteritis -34 Virus (EAV). Most preferably, the virus is HIV-1, HIV-2, the SARS virus, Coronaviruse 229E, Coronavirus OC43, PRCV, BCV, HCV, or EAV. Other Coronaviruses which can be inhibited or their infections treated by the compounds of the invention are those listed in Table 1. 5 According to an embodiment, there is provided a method for preventing the infection of a cell exposed to a virus comprising contacting said cell with a compound according to the embodiemnts. Preferably, the virus is from the Lentivirus family, or the Coronavirus family. More preferably, the virus is Human Immunodeficiency Virus (HIV), Severe Respiratory 10 Syndrome virus (SARS), Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus (BCV), Porcine Respiratory Coronavirus (PRCV), Mouse Hepatitis virus (MHV), Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Most preferably, the virus is HIV-1, HIV-2, the SARS virus, Coronaviruse 229E, Coronavirus OC43, PRCV, BCV, HCV, EAV. 15 Other Coronaviruses which can be inhibited or their infections treated by the compounds of the invention are those listed in Table 1. According to an embodiment of the invention, there is provided a method for the therapeutic or prophylactic treatment of a subject infected with or exposed to a virus, comprising the administration of a compound according to the embodiments, to a subject 20 in need of said treatment. Preferably, infection with a virus or exposure to a virus occurs with viruses belonging to the Lentivirus family, or the Coronovirus family. More preferably, infection or exposure occurs with HIV, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus (BCV), Porcine 25 Respiratory Coronavirus (PRCV), Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Most preferably, infection or exposure occurs with HIV-1, HIV-2, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Other Coronaviruses which can be inhibited or their infections treated by the 30 compounds of the invention are those listed in Table 1. The subject of the viral inhibition is generally a mammal such as but not limited to human, primate, livestock animal (e.g. sheep, cow, horse, donkey, pig), companion animal (e.g. dog, cat), laboratory test animal (e.g. mouse, rabbit, rat, guinea pig, -35 hamster), captive wild animal (e.g. fox, deer). Preferably, the subject is a primate, or horse. Most preferably, the subject is a human. According to an embodiment, there is provided a method of down regulating a membrane ion channel functional activity in a cell infected with a virus, comprising 5 contacting said cell with a compound according to the embodiments. The membrane ion channel may be endogenous to the cell or exogenous to the cell. Preferably, the membrane ion channel of which functional activity is down regulated is that which Lentiviruses, and Coronaviruses utilise for mediating viral replication and include, for example, the HIV membrane ion channel Vpu , the HCV 10 membrane ion channel P7, the Coronavirus E protein membrane ion channel, and the SARS E protein membrane ion channel. Preferably, infection with a virus or exposure to a virus occurs with viruses belonging to the Lentivirus family, or the Coronovirus family. More preferably, infection or exposure occurs with HIV, SARS, Human Coronavirus 229E, Human 15 Coronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus (BCV), Porcine Respiratory Coronavirus (PRCV), Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Most preferably, infection or exposure occurs with HIV-I, HIV-2, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). 20 According to an embodiment of the present invention, there is provided a method of reducing, retarding or otherwise inhibiting growth and/or replication of a virus that has infected a cell, said method comprising contacting said infected cell with a compound according to the embodienmts, wherein said compound down regulates functional activity of a membrane ion channel derived from said virus and expressed in said 25 infected cell. Preferably, infection occurs with a virus belonging to the Lentivirus family, or the Coronovirus family. More preferably, infection or exposure occurs with HIV, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus (BCV), Porcine Respiratory Coronavirus (PRCV), Hepatitis C virus 30 (HCV), or Equine Arteritis Virus (EAV). Most preferably, infection or exposure occurs with HIV-1, HIV-2, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV).

-36 Other Coronaviruses which can be inhibited or their infections treated by the compounds of the invention are those listed in Table 1. Preferably, the membrane ion channel of which functional activity is down regulated is that which Lentiviruses, and Coronaviruses utilise for mediating viral 5 replication and include, for example, the HIV membrane ion channel Vpu , the HCV membrane ion channel P7, and the Coronavirus E protein membrane ion channel. According to an embodiment, the present invention provides a method of reducing, retarding or otherwise inhibiting growth and/or replication of a virus that has infected a cell in a mammal, said method comprising administering to said mammal a compound 10 according to the embodiments, or a pharmaceutical composition according to the embodiments, wherein said compound or said composition down regulates functional activity of a membrane ion channel expressed in said infected cell. Preferably, infection occurs with a virus belonging to the Lentivirus family, or the Coronovirus family. More preferably, infection or exposure occurs with HIV, SARS, 15 Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus (BCV), Porcine Respiratory Coronavirus (PRCV), Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Most preferably, infection or exposure occurs with HIV-1, HIV-2, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). 20 Other Coronaviruses which can be inhibited or their infections treated by the compounds of the invention are those listed in Table 1. Preferably, the membrane ion channel of which functional activity is down regulated is that which Lentiviruses, and Coronaviruses utilise for mediating viral replication and include, for example, the HIV membrane ion channel Vpu , the HCV 25 membrane ion channel P7, and the Coronavirus E protein membrane ion channel. The subject of the viral inhibition is generally a mammal such as but not limited to human, primate, livestock animal (e.g. sheep, cow, horse, donkey, pig), companion animal (e.g. dog, cat), laboratory test animal (e.g. mouse, rabbit, rat, guinea pig, hamster), captive wild animal (e.g. fox, deer). Preferably, the subject is a primate, or 30 horse. Most preferably, the subject is a human. According to an embodiment, the present invention provides a method for the therapeutic or prophylactic treatment of a subject infected with or exposed to a virus comprising administering to said subject a compound according to the embodiments, or -37 a pharmaceutical composition according to the embodiments, wherein said compound or said composition down-regulates functional activity of a membrane ion channel derived from said virus. Preferably, infection occurs with a virus belonging to the Lentivirus family, or the 5 Coronovirus family of viruses. More preferably, infection or exposure occurs with HIV, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus (BCV), Porcine Respiratory Coronavirus (PRCV), Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Most preferably, infection or exposure occurs with HIV-1, HIV-2, SARS, Human Coronavirus 229E, Human 10 Coronavirus OC43, Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Other Coronaviruses which can be inhibited or their infections treated by the compounds of the invention are those listed in Table 1. Preferably, the membrane ion channel of which functional activity is down regulated is that which Lentiviruses, and Coronaviruses utilise for mediating viral 15 replication and include, for example, the HIV membrane ion channel Vpu , the HCV membrane ion channel P7, and the Coronavirus E protein membrane ion channel,. The subject of the viral inhibition is generally a mammal such as but not limited to human, primate, livestock animal (e.g. sheep, cow, horse, donkey, pig), companion animal (e.g. dog, cat), laboratory test animal (e.g. mouse, rabbit, rat, guinea pig, 20 hamster), captive wild animal (e.g. fox, deer). Preferably, the subject is a primate, or horse. Most preferably, the subject is a human. According to an embodiment, the invention provides an antiviral compound selected from the group consisting of: N-(3,5-Diamino-6-chloro-pyrazine-2-carbonyl)-N'-phenyl-guanidine, 25 N-Benzyl-N'-(3,5-diamino-6-chloro-pyrazine-2-carbonyl)-guanidine, 3'4 DichloroBenzamil, 2'4 DichloroBenzamil, 5-(N-methyl-N-guanidinocarbonyl-methyl)amiloride, 5-(N-Methyl-N-isobutyl)amiloride, 30 5-(N-Ethyl-N-isopropyl)amiloride, 5-(N,N-Dimethyl)amiloride hydrochloride, 5-(N,N-hexamethylene)amiloride, 5-(N,N-Diethyl)amiloride hydrochloride, -38 6-lodoamiloride, Bodipy-FL amiloride, 3-hydroxy-5-hexamethyleneimino-amiloride, 5-(4-fluorophenyl)amiloride, 5 5 -tert-butylam ino -amilo ride, N -amid ino -3 -am ino-5 -pheny 1-6-c hlo ro-2-pyrazinecarboxam ide, 3-methoxy-5 -(N,N-H-examethylene)-amiloride, 3 -methoxy-ami lo ride, hexamethyleneimino-6-phenyl-2-pyrazinecarboximide, 1 0 N-amidino-3,5-diamino-6-phenyl-2-pyrazinecarboxamide, 1 -naphthoylguanidine, 2-naphthoylguanidine, N-(2-naphthoyl)-N'-phenylguanidine, N,N'-bis(2-naphthoyl)guanidine, 15 N,N'-bis( I -naphthoyl)guanidine, N,N'-bis(2-naphthoyl)-N"-phenylguanidine, 6-methoxy-2-naphthoylguanidine, 3-quinolinoylguanidine, cinnamoylguanidime, 20 4-phenylbenzoylguanidine, WO 2004/112687 PCT/AU2004/000866 -39 N-(cinnamoyl)-N'phenylguanidine, (3-phenylpropanoyl)gianidine, N,N'-bis-(cinnamoyl)-N'-phenylguanidine, N-(3-phenylpropanoyl)-N-phenylguanidine, N,N-bis(3phenylpropanoyl)-N"-phenyluanidine, trans-3-furanacryoylguanidine, N-(6-Hydroxy-2-naphthoyl)-N'-phenylgnanidine (4-Phenoxybenzoyl)guanidine, NN-Bis(amidino)napthalene-2,6-dicarboxamide, 10 N"-Cinnamoyl-NN'-diphenylguanidine, (Phenylacety)guanine, N,N'-Bis(3-phenylpropanoyl)guanidine, benzoylguanidine, (4-Chlorophenoxy-actyl]guanidine 15 N-benzoyl-N-cinnamoylguanidine, [(E)-3-(4-Dimethylaminopheny1)-2-methylacryloy1]guanidine, (4-Chlorocinnamoyl)guanidine, (4-Bromocinnamoy1)guanidine, (4-Methoxycinnamoyl)guanidine, 20 (5-Phenyl-penta-2,4-dienoyl)guanidine, (3-Bromocinnamoyl)guanidine, (3-Methoxycinnamoyl)guanidinc, (3-Chlorocinnamoyl)guanidine, (2-Chlorocinnamoyl)guanidine, 25 (2-Bromocinnamoyl)guanidine. (2-Methoxycimiamoyl)guanidine, (trans-2-Pheny1cyclopropanecarbonyl)guanidine, [3-(3-Pyridyl)acryloyl]guanidine, (4-Hydroxycinmamoyl)guanidine, 30 (Quinoline-2-carbonyl)guanidine, or pharnaceutically acceptable salts thereof.

-40 According to an embodiment, the present invention provides a pharmaceutical composition comprising a compound according to the embodiments, and optionally one or more pharmaceutical acceptable carriers or derivatives. Preferably, the pharmaceutical composition may further comprise one or more 5 known antiviral compounds or molecules. Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to". 10 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic representation of plasmids used for expression of Vpu in E. coli. A. The amino acid sequence ( < 400 > 1) encoded by the vpu open reading frame (ORF) 15 generated by PCR from an HIV- 1 strain HXB2 cDNA clone. The vpu ORF was cloned in-frame at the 3' end of the GST gene in p2GEX to generate p2GEXVpu (B). It was subsequently cloned into pPL451 to produce the plasmid pPL + Vpu (C). Figure 2 is a photographic representation of the expression and purification of Vpu in E. 20 coli. A. Western blotting after SDS-PAGE was used to detect expressed Vpu in E. coli extracts. Lanes 1-4 contain samples, at various stages of purity, of Vpu expressed from p2GEXVpu: lane 1, GST-Vpu fusion protein isolated by glutathione-agarose affinity chromatography; lane 2, Vpu liberated from the fusion protein by treatment with thrombin; lane 3, Vpu purified by HPLC anion exchange chromatography; lane 4, Vpu 25 after passage through the immunoaffinity column. Lanes 5 and 6, membrane vesicles prepared from 42'C induced cells containing pPL+Vpu or pPL45 1, respectively. B. Silver stained SDS-PAGE gel: lane 1, Vpu purified by HPLC anion exchange chromatography; lane 2, Vpu after passage through the immunoaffinity column. 30 Figure 3 is a graphical representation of ion channel activity observed after exposure of lipid bilayers to aliquots containing purified Vpu. In A and B, the CIS chamber contained 500mM NaCl and the TRANS chamber contained 50mM NaCl; both WO 2004/112687 PCT/AU2004/000866 -41 solutions were buffered at pH 6.0 with 10 mM MES. B shows a current versus voltage curve generated from data similar to that shown in A. Figure 4 is a photographic representation of bacterial cross-feeding assays. For all plates, the Mof, Pro" auxotrophic strain was used to seed a soft agar overlay. Plates A 5 and B contain minimal drop-out medium minus proline; in plate C the medium was minus methionine. To control for viability of the cells in the background lawn, the discs labelled F and M contained added proline or methionine, respectively. The discs labelled C and V were inoculated with Met Pro E, cohl cells containing the plasmids pPL451 or pPL+Vpu, respectively. Plates were incubated at 37*C (A and C) 10 or 30*C (B) for two days and photographed above a black background with peripheral illumination from a fluorescent light located below the plate. The images were recorded on a Novaline video gel documentation system. Light halos around the discs labelled P or M on all plates and around the disc labelled V on plate A indicate growth of the background lawn strain. 15 Figure 5 is a graphical representation of the screening of drugs for potential Vpu channel blockers. The photograph shows a section of a minimal medium-lacking adenine - agarose plate onto which a lawn of XL-I-blue E. col cells containing the Vpu expression plasmid pPLVpu has been seeded. Numbers 6-11 are located at the sites of application of various drugs being tested, which were applied in 3pl drops and 20 allowed to soak into the agarose. The plate was then incubated at 370C for 48hr prior to being photographed. The background grey shade corresponds to areas of no bacterial growth. The bright circular area around " 10 " represents bacterial cell growth as a result of application of adenine at that location (positive control). The smaller halo of bacterial growth around "9" is due to the application of 5-(NN 25 hexamethylene)-amiloride at that location. Figure 6. SARS B protein ion channel activity observed in NaCl solutions after exposure of lipid bilayer to 3-1Og of E protein. A. The closed state is shown as solid line, openings are derivations from the line. Scale bar is 3OOms and SpA. The CIS 30 chamber contained 50mM NaCl in 5mM HEPES buffer pH 7.2, the TRANS chamber contained 500mM NaCl in SmM IJEPES buffer pH 7.2. The CIS chamber was.

WO 2004/112687 PCT/AU2004/000866 -42 earthed and the TRANS chamber was held at various potentials between -100 to +1 00mV. B. Largest single opening events of a single channel. Figure 7. SARS E protein ion channel activity observed in NaCi solutions after 5 exposure of lipid bilayer to 3-10lg of E protein. A. The closed state is shown as solid line, openings are derivations from the line. Scale bar is 300ms and SpA. The CIS chamber contained 50mM NaCl in 5mM HEPES buffer pH 7.2, the TRANS chamber contained 500mM NaCI in SmM HEPES buffer pH 7.2. The CIS chamber was earthed and the TRANS chamber was held at various potentials between -100 to 10 +10mV. B. Largest single opening events of a single channel. Figure 8. Cinnamoylguanidine (Bit036) inhibits SARS E protein ion channel activity in.NaCi solution. A. Representative currents at holding potential of-40mV, Scale bar is 300mS and 5pA. E protein ion channel activity and E protein channel activity after the addition of 100sM BitO36. B. All points bistogram at holding potential of 15 40mV. E protein ion channel activity before and after the addition of 100pM BitO36. C. Average current (pA), before formation of E protein ion channel, E protein ion channel activity and after addition of I OOpM Bit036. Figure 9 229E E protein Ion channel activity in lipid bilayers in K1 solutions. Figure 10: Part A shows raw currents generated by the 229E-E protein ion channel 20 in a planar lipid bilayer.. The top trace shows current activity prior to drug addition and the lower trace shows the effect of addition of 100pM cinnamoylguanidine on channel activity. Part B is a graphical representation of the average current flowing across the bilayer (in arbitrary units), before and after addition of cinnamoylguanidine. 25 Figure 11: MHV E protein Ion channel activity in lipid bilayers NaCJ solutions. Figure 12: Part A shows raw currents generated by the MHV-E protein ion channel in a planar lipid bilayer. The top trace shows current activity prior to drug addition and the lower trace shows the effect of addition of 100pM cinniamoylguanidine on 30 channel activity, Part B is a graphical representation of the average current flowing WO 2004/112687 PCT/AU2004/000866 -43 across the bilayer (in arbitrary units), before and after addition of cinnamoylguanidine. DETAILED DESCRIPTION OF THE INVENTION The present invention is based, in part, on the surprising determination that 5 certain compounds that fall under the classification of substituted acylguanidines have antiviral activity against viruses from a range of different virus families. Without intending to be bound by any particular theory or mechanism of action, the negative impact of the compounds of the present invention on viral replication may be mediated by the inhibition or otherwise down-regulation of a membrane ion channel 10 relied upon by the virus for replication. This membrane ion channel may be a viral membrane ion channel (exogenous to the host cell) or a host cell ion channel induced as a result of viral infection (endogenous to the host cell). As an example, the compounds of the present invention may inhibit Vpu or p7 function and thereby inhibit the continuation of the respective HIV or HCV life cycle. 15 The SARS virus encodes an E protein which is shown for the first time, by the present inventors, to act as an ion channel. As similar B proteins are present in other coronaviruses, the compounds, compositions and methods of the present invention would have utility in the inhibition and/or treatment of infections by other coronaviruses. 20 While the present invention is concerned with novel antiviral compounds falling under the classification of substituted acylguanidines, it does not include in its scope the use of compounds 5-(NN-hexamethylene)amiloride and 5-(N,N-dimethyl) amiloride for retarding, reducing or otherwise inhibiting viral growth and/or functional activity of HI V. 25 It will be understood by those skilled in the art that the compounds of the invention may be administered in the- form of a composition or formulation comprising pharmaceutically acceptable carriers and excipients. The phatmacentical compositions of the invention may further comprise one or more known antiviral compounds or molecules. Preferably, the known antiviral 30 compounds are selected from the group consisting of Vidarabine, Acyclovir, Ganciclovir, Valganciclovir, Valacyclovir, Cidofovir, Famciclovir, Ribavirin, WO 2004/112687 PCT/AU2004/000866 Amantadine, Rimantadine, Interferon, Oseltamivir, Palivizunab, Rimantadine, Zanamivir, nucleoside-analog reverse transcriptase inhibitors (NRTI) such as Zidovudine, Didanosine, Zalcitabine, Stavudine, Lamivudine and Abacavir, non nucleoside reverse transcriptase inhibitors (NNRTI) such as Nevirapine, Delavirdine 5 and Efavirenz, protease inhibitors such as Saquinavir, Ritonavir, Indinavir, Nelfmavir, Amprenavir, and other known antiviral compounds and preparations. Known antiviral compounds or molecules may in some cases act synergistically with the antiviral compounds of the invention. 10 Table 1 Known coronavirus Isolates Group I species Canine coronavirus Canine enteric coronavims (strain INSAVC-1) Canine enteric coronavirus (strain K378) Feline coronavirus Feline enteric coronavirus (strain 79-1683) Feline infectious peritonitis virus FI Human comonavims 229E Porcine epidemic diarrhea virus Porcine epidemic diarhea virus (strain Br1/87) Porcine epidemic diarrhea virus (strain CV777) Transmissible gsretrtsvirus Porcine respirtor coronavirus Porcine transmissible gastroenteritis coronavirus (STRAIN FS772/70) Porcine transmissible gastroenteritis coronavirus (strain Miller) Porcine transmissible gastroenteritis coronavirus (strain Neb72-RT) Porcine transmissible gastroenteritis coronavirus (STRAIN PURDUE) Group 2 species Bovine coronavirus Bovine coronavirus (STRAIN F15) Bovine coronavirus (strain G95) Bovine coronavirus (STRAIN L9) Bovine coronavirus (strain LSU-94LSS-051) Bovine coronavirus (STRAIN LY-138) Bovine coronavirus (STRAIN MEBUS) Bovine coronavirus (strain OK-0514-3) Bovine coronavimus (stain Ontario)_ Bovine coronavirus (STRAIN QUEBEC) Bovine coronavirus (STRAIN VACCINE) Bovine enteric coronavirus (strain 98TXSF-I 10-ENT) Canine respirator coronavirus WO 2004/112687 PCT/AU2004/000866 -45 Chicken enteric coronavirus Human coronavirus OC43 Murine hepatitis virus Murine coronavirus (strain DVEM) Murine hepatitis virus (strain A59) Murine hepatitis virus (strain JHM) Murine hepatitis virus (strain S) Murine hepatitis virus strain 1 Murine hepatitis virus strain 2 Murine hepatitis virus strain 3 Murine hepatitis virs strain 4 Murine hepatitis virus strain ML-1 1 Porcine hemagglutinating encephalomyelitis virus Porcine hemagglutinating encephalomyelitis virus (strain 67N) Porcine hemagglutinating encephalomyelitis virus (strain IAF-404) Puffinosis virus Rat coronavirus Rat coronavirus (strain 681) Rat coronavirus (strain NJ) Rat sialodacryoadenitis coronavirus Group 3 species Turkey coronavirus Turkey coronavims (stra Indiana) Turke coronavirus (strain Minnesota) Turkey coronavirus (strain NC95) Avian infectious bronchitis virus Avian infectious bronchitis virus (STRAIN 6/82) Avian infectious bronchitis virus (strain Arkansas 99) Avian infectious bronchitis virus (strain Beaudette ) Avian infectious bronchitis virus (strain Beaudete M42) Avian infectious bronchitis vims (strain Beaudette US) Avian infectious bronchitis virus (strain Beaudee) Avian infectious bronchitis virus (strain D1466) Avian infectious bronchitis virus (strain D274) Avian infectious bronchitis virus (strain D3896) Avian infectious bronchitis vims (strain D41) Avian infectious bronchitis virus (strain DE072) Avian infecious bronchitis viru (atrai GRAY) Avian infectious bronchitis virus (stai H120) Avian infectious bronchitis virus (strain H52) Avian infectious bronchitis virus (strain KB8523). Avian infectious bronchitis virus (strain M41) Avian infectious b-ronichitis virus straini PORTUGAL/322/82) Aian infectious bronchitis virus (strain SAIB20) WO 2004/112687 PCT/AU2004/000866 A46 Avian infectious bronchitis virus (strain UK/123/82) Avian infectious bronchitis virus (strain UK/I 421/86) Avian infectious bronchitis virus (strain UK/167684) Avian infectious bronchitis virus (strain UK/I 83/66) Avian infectious broncbitis virus (strain K/68/84) Avian infectious bronchitis virus (strain Vic S) Avian infectiosarntrceisvm Preliminary Group 4 species SARS coronavirus SARS coronavirus Bijing ZY-2603 SARS coronavirus BJO1 SARS coronavirus B102 SARS coronavhus BJ03 SARS coronavius BJ04 SARS coronaviras CUHK-S10 SARS coronavirus CU- W1 SARS coronavirus Frankfu-t I SARS coronavirus GZ01 SARS coronavirus HKU-39849 SARS coronavirus Hn Kong ZY-2003 SARS coronavirus Hong Ko /2003 SARS coronavirus iSR SARS coronavirus Sin2500 SARS coronavirus Sin2677 SARS coronavirus Sin2679 SARS coronavirus Sin2748 SARS coronavirus Sin2774 SARS coronavihs Taiwan SARS coronavirs Taiwan JC-2003 SARS coronavirus Taiwan C SARS coronavirus TaiwanTC2 SARS coronavirus Tor2 SARS coronavirus TW I SARS coronavims TWC SARS coronavims Urbani SARS coronavirus Vietnam SARS coronavims ZJ-HZ01 SARS coronavirus zJ01 unclassified coronaviruses Bovine respiratory coronavirus (strain 98TXSF-I I 0-LUN) Human enteric coronavirus 4408 Enteric coronavirus Equine coronavitus WO 2004/112687 PCT/AU2004/000866 -47 Equine coronavirus NC99 The present observations and findings now permit the use of agents such as certain substituted acylguanidines, as anti-viral agents for the therapy and prophylaxis of viral conditions caused by different viruses. The methods and compositions of the 5 present invention may be particularly effective against viruses which rely on ion channel formation fbr their replication, however it will be understood that this is not the only mechanismrelied on by viruses for replication and that the compounds and methods of the present invention are not limited to agents which exert their action by retarding or inhibiting the function of ion channels. 10 Reference to."membrane ion channel" should be understood as a reference to a structure which transports ions across a membrane. The present invention extends to ion channels which may function by means such as passive, osmotic, active or exchange transport, The ion channel may be formed by intracellular or extracellular means. For example, the ion channel may be an ion channel which is naturally formed 15 by a cell to facilitate its normal functioning. Alternatively, the ion channel maybe formed by extracellular means. Extracellular means would include, for example, the formation of ion channels due to introduced chemicals, drugs or other agents such as ionophores or due to the functional activity of viral proteins encoded by a virus which has entered a cell. 20 The ion channels which are the subject of certain embodiments of the present invention facilitate the transport of ions across membranes. Said membrane may be any membrane and is not limited to the outer cell wall plasma membrane. Accordingly, "membrane" as used herein encompasses the membrane surrounding any cellular organelle, such as the Golgi apparatus and endoplasmic reticulum, the 25 outer cell membrane, the membrane surrounding any foreign antigen which is located within the cell (for example, a viral envelope) or the membrane of a foreign organism which is located extracellularly. The membrane is typically, but not necessarily, composed of a fluid lipid bilayer. The subject ion channel maybe of any structure. For example, the Vpu ion channel is formed by Vpu which is an integral membrane 30 protein encoded by HW-1 which associates with, for example, the Golgi and endoplasmic reticulum membranes of infected cells. Reference hereinafter to "Vpu WO 2004/112687 PCT/AU2004/000866 -48 ion channels" is a reference to all related ion channels for example P7 HCV and M2 of influenza and the like. Reference to "lIV", "SARS", "Coronavirus" or "HTCV" should be understood as a reference to any HV, SARS, Coronavirus or HCV virus strain and including 5 homologues and mutants. Reference to the "functional activity" of an ion channel should be understood as a reference to any one or more of the functions which an ion channel performs or is involved in. For example, the Vpu protein encoded ion channel, in addition to facilitating the transportation ofNat , cr and P 4 3, also plays a role in the 10 degradation of the CD4 molecule in the endoplasmic reticulum. Without wishing to be bound by a particular theory, the Vpu protein encoded ion channel is also thought to play a role in mediating the HIV life cycle. The present invention is not limited to treating HIV infection via the mechanism of inhibiting the HIV life cycle and, in particular, HIV replication. Rather, the present invention should be understood to 15 encompass any mechanism by which the compounds of the present invention exert their anti-viral activity and may include inhibition of II1V viability or functional activity. This also applies to HCV, Coronaviruses, and to other viruses. Reference to the "functional activity" of a virus should be understood as a reference to any one or more of the functions which a virus performs or is involved 20 in. Reference to the " viral replication" should be understood to include any one or more stages or aspects ofthe viral life cycle, such as inhibiting the assembly or release of virious. Ion channel mediation of.viral replication may be by direct or indirect means. Said ion channel mediation is by direct means if the ion channel 25 interacts directly with the virion at any one or more of its life cycle stages. Said ion channel mediation is indirect if it interacts with a molecule other than those of the virion, which other molecule either directly or indirectly modulates any one or more aspects or stages of the viral life cycle. Accordingly, the method of the present invention encompasses the mediation of viral replication via the induction of a 30 cascade of steps which lead to the mediation of any one or more aspects or stages of the viral life cycle.

WO 2004/112687 PCT/AU2004/000866 -49 Reference to "down-regulating' ion channel functional activity, should be understood as a reference to the partial or complete inhibition of any one or more aspects of said activity by both direct and indirect mechanisms. For example, a suitable agent may interact directly with an ion channel to prevent replication of a 5 virus or, alternatively, may act indirectly to prevent said replication by, for example, interacting with a molecule other than an ion channel. A further alternative is that said other molecule interacts with and inhibits the activity of the ion channel. Screening for molecules that have antiviral activity can be achieved by the range of methodologies described herein. 10 Reference to a "cell" infected with a virus should be understood as a reference to any cell, prokaryotic or eukaryotic, which has been infected with a virus. This includes, for example, immortal or primary cell lines, bacterial cultures and cells in situ. In a suitable screening system for antiviral compounds, the preferred infected cells would be macrophages/monocytes or hepatocytes/Iymphoid cells infected with 15 either HIV or HCV respectively. Without limiting the present invention to any one theory or mode of action, The compounds of the present invention are thought to inhibit viral replication or virion release from cells by causing ion channels, namely VPU of HIV, the E protein of SARS and other Coronaviruses, or P7 of HCV to become blocked. The present 20 invention encompasses antiviral compounds that are substituted acylgaanidines. The present invention also includes the use of compounds 5-(NN hexamethylene)amiloride and 5-(N,N-dimethyl)-amiloride in the control of viral replication and/or growth other than HIV. The subject of the viral inhibition is generally a mammal such as but not 25 limited to human, primate, livestock animal (e.g. sheep, cow, horse, donkey, pig), companion animal (e.g. dog, cat), laboratory test animal (e.g. mouse, rabbit, rat, guinea pig, hamster), captive wild animal (e.g. fox, deer). Preferably, the subject is a human or primate. Most preferably, the subject is a human. The method of the present invention is useful in the treatment and prophylaxis 30 of viral infection such as, for example, but not limited to IV infection, HCV infection and other viral infections. For example, the antiviral activity may be effected in subjects known to be infected with IV in order to prevent replication of WO 2004/112687 PCT/AU2004/000866 HIV thereby preventing the onset of AIDS. Alternatively, the method of the present invention may be used to reduce serum viral load or to alleviate viral infection symptoms. Similarly, antiviral treatment may be effected in subjects known to be infected with, for example, HCV, in order to prevent replication of HCV, thereby 5 preventing the father hepatocyte involvement and the ultimate degeneration of liver tissue. The method of the present invention may be particularly useful either in the 'early stages of viral infection to prevent the establishment of a viral reservoir in affected cells or as a prophylactic treatment to be applied immediately prior to or for 10 a period after exposure to a possible source of virus, Reference herein to "therapeutic" and "prophylactic" is to be considered in their broadest contexts. The term "therapeutic' does not necessarily imply that a mammal is treated until total recovery. Similarly, "prophylactic" does not necessarily mean that the subject will not eventually contract a disease condition. Accordingly, 15 - therapy and prophylaxis include amelioration. of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition. The term "prophylaxis"may be considered as reducing the severity of onset of a particular condition. Therapy may also reduce the severity of an existing condition or the frequency of acute attacks, 20 In accordance with the methods of the present invention, more than one compound or composition may be co-administered with one or more other compounds, such as known anti-viral compounds or molecules. By "co administered" is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by 25 the same or different routes. By "sequential" administration is meant a time difference of from seconds, minutes, hours or days between the administration of the two or more separate compounds. The subject antiviral compounds may be administered in any order. Routes of administration include but are not limited to intravenously, 30 intraperitionealy, subcutaneously, intracranialy, intradermally, intramuscularly, intraocularly, intrathecaly, intracerebrally, intranasally, transmucosally, by infusion, WO 2004/112687 PCT/AU2004/000866 -51 orally, rectally, via iv drip, patch and implant. Intravenous routes are particularly preferred. Compositions suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of 5 sterile injectable solutions. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, 10 phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, fbr example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active 15 compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by, for example, filter sterilization or sterilization by other appropriate means. Dispersions are also contemplated and these may be prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the 20 required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, a preferred method of preparation includes vacuum drying and the freeze-drying teolmique which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution. 25 When the active ingredients are suitably protected, they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, 30 trocbes, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.01 % by weight, more preferably 0.1% by weight, even more preferably 1% by weight of active compound.

WO 2004/112687 PCT/AU2004/000866 -52 The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 1 to about 99%, more preferably about 2 to about 90 %, even more preferably about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a 5 suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 ng and 2000 mg of active compound. The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: A binder such as gum, acacia, corn starch or gelatin; 10 excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a 1$ liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Any material 20 used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound(s) may be Incorporated into sustained-release preparations and formulations. The present invention also extends to forms suitable for topical application such as creams, lotions and gels. In such fbrms, the anti-clotting peptides may need to 25 be modified to permit penetration of the surface barrier. Procedures for the preparation of dosage unit forms and topical preparations are readily available to those skilled in the art from texts such as Pharmaceutical Handbook. A Martinddle Companion Volume Ed. Ainley Wade Nineteenth Edition The Pharmaceutical Press London, 30 CRC Handbook of Chemistry and Physics Ed. Robert C. East Ph D. CRC Press Inc.; Goodman and Gilman's; The Pharmacological basis of Therapeutics. Ninth Ed WO 2004/112687 PCT/AU2004/000866 -53 McGraw Hill; Remington; and The Science and Practice of Pharmacy. Nineteenth Ed. Ed. Alfonso R. Gennaro Mack Publishing Co. Easton Pennsylvania. Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and 5 absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. 10 It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated;'cach unit containing a predetermined quantity of active material calculated.to produce the desired therapeutic effect in association with 15 the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved and (b) the limitations inherent in the art of compounding. Effective amounts contemplated by the present invention will vary depending 20 on the severity of the pain and the health and age of the recipient. In general terms, effective amounts may vary from 0.01 ng/kg body weight to about 100 mg/kg body weight. Alternative amounts include for about 0. 1 ng/kg body weight about 100 mg/kg body weight or from 1.0 ng/kg body weight to about 80 mg/kg body weight. 25 The subject of the viral inhibition is generally a mammal such as but not limited to human, primate, livestock animal (e.g. sheep, cow, horse, donkey, pig), companion animal (e.g. dog, cat), laboratory test animal (e.g. mouse, rabbit, rat, guinea pig, hamster), captive wild animal (e.g. fox, deer). Preferably, the subject is a human or primate. Most preferably, the subject is a human. 30 The methods of the present invention is useful in the treatment and prophylaxis of viral infection such as, for example, but not limited to H[V infection, HCV infection and other viral infections. For example, the antiviral activity may be.

WO 2004/112687 PCT/AU2004/000866 -54 effected in subjects known to be infected with HIV in order to prevent replication of HIV thereby preventing the onset of AIDS. Alternatively, the methods of the present invention may be used to reduce serum viral load or to alleviate viral infection symptoms. Similarly, antiviral treatment may be effected in subjects known to be 5 infected with, for example, HCV, in order to prevent replication of HCV, thereby preventing the ftrther hepatocyte involvement and the ultimate degeneration of liver tissue. The methods of the present invention maybe particularly useful either in the early stages of viral infection to prevent the establishment of a viral reservoir in 10 affected cells or as a prophylactic treatment to be applied immediately prior to or for a period after exposure to a possible source ofvirus. The present invention will now be described in more detail with reference to specific but non-limiting examples describing studies of viral membrane ion channels and screening for antiviral activity. Some examples involve the use of the SARS 15 virus. It will be clear from the description herein that other lentiviruses, and coronaviruses and other compounds may be used effectively in the context of the present invention. It is to be understood, however, that the detailed description is included solely for the purpose of exemplifying the present invention. It should not be understood in any way as a restriction on the broad description of the invention as set 20 out above. Example . Synthesis of the Compounds of the Invention. The compounds of the present invention may be made from the corresponding acid oblorides or methyl esters as shown in Scheme 1. Both of these methods are well described in the literature. 0 0 NH 0 R O01 R N R OMe acid chlodde acylgaaidine esTer Sche= I 25 WO 2004/112687 PCT/AU2004/000866 -55 The following examples show synthetic schemes for some compounds of the invention. Example 2. Synthesis of Cinnamovlpuanidine from Cinnamic acid Cinnamovl chloride 5 0 L (COCi>.Ibw~uhnLDNW NI .2.(H2N)j=NlHQI NH NaOH 1110 /THF To a solution of trans-cinnamic acid (1.50 g, 10. 12 mmol) in dry benzene (30mL) containing a drop of N,N-dimethylformamide was added oxalyl chloride .10 (5.14 g, 40.5 mmol) causing the solution to effervesce. Afke refluxing for 2 h, the solution was evaporated to dryness under reduced pressure. The resulting solid was dissolved in dry tetrahydrofuran (2OmL) and added slowly to a solution of guanidine hydrochloride in 2M aqueous sodium hydroxide (25mL). The reaction was stirred at room temperature for lb then extracted with ethyl acetate (3x5OmL). The combined 15 extracts were dried over magnesium sulfate and evaporated to give an orange oil. The crude product was purified by column chromatography. Elution with 10% to 20% methanol in dichloromethane gave Cinnamoylguanidine as a cream solid (0.829g 43%). Example 3 20 Synthesis of N-amidino-3-amino-5-phenvi-6-chloro-2-pvrazinecarboxamide. Part 1 H2 To a solution of methyl 3-amino-5,6-dichloro-2-pyrazinecearboxylate (0.444 g, 2.0 mmol) in tetrahydrofbran (5 mL) / water (10 mL) / toluene (20 mL) was added 25 phenyl boronic acid (0.536 g, 4.4 mmol), sodium carbonate (0.699 g, 6.6 mmol) and tetrakis(triphenylphosphine)- palladium(O) (0.116 g, 0.10 nmol). The reaction was WO 2004/112687 PCT/AU2004/000866 -56 evacuated and purged with nitrogen several times before being refluxed for 6 h. The organic layer was separated and the aqueous layer extracted with toluene (3 x 20 mL). The combined organic extracts were dried over magnesium sulfate, filtered and evaporated under reduced pressure to give methyl 3-amino-6-chloro-S 5 phenyl-2-pyrazinecarboxylate as a yellow solid (0.43 g, 82%). Part 2 K.mtanol H2

NH

2 To a solution of sodium (0.040 g, 174 mmol) dissolved in methanol (5 mL) 10 was added guanidine hydrochloride (0.258 g, 2.70 mmol) and the mixture refluxed for 30 min after which it was filtered. To the filtrate was added methyl 3-amino-6 cbloro-5-pheny1-2-pyrazinecarboxylate (0.264 g, 1.0 mmol) in NN dimethylformamide (5 mL) and the solution heated at 75 0 C for 12 h. The solvent was removed under reduced pressure and the residue chromatographed on silica gel 15 eluting with 1% triethylamine / 5% methanol / dichloromethane. The resulting solid was suspended in chloroform, filtered and dried under high vacuum to give N Amidino-3-amino-5-phenyl--chloro-2-pyrazinecarboxamide as a yellow solid (0.04 g, 14%). Example 4. 20 Synthesis of hexamethyleneimino-6-phenyl-2-pyrazinecarboxamide Part I 0 + CA N NH2 N NH2 To a solution of methyl 3-amino-5,6-dichloro-2-pyrazinecarboxylate (1,11 g, 5.0 mmol) in tetrahydrofuran (50 mL) was added hexamethyleneimine (1.49 g, 15.0 25 mmol) and the reaction was refluxed for I h. The reaction was allowed to cool and the solid hexamethyleneimine hydrochloride removed by filtration. The filtrate was WO 2004/112687 PCT/AU2004/000866 -57 evaporated and the residue chromatographed over silica gel. Elution with dichloromethane gave methyl 3-amino-6-chloro-5-hexamethyleneimino-2 pyrazinecarboxylate as an off-white solid (1.20 g, 85%). 5 Part2 DMS IM KpC6 PdC3GippO To a solution of methyl 3-amino-6-chloro-5-hexamethyleneimino-2 pyrazinecarboxylate (0.350g, 1.23 mmol) in dimethylsulfoxide (5 mL) was added phenyl boronic acid (0.166 g, 1.35 mmol), potassium carbonate (0.511 g, 3.70 mmol) 10 and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(H)-dichloromethane complex (0.041 g, 0.05 mmol). The reaction was heated at 90.C fbr 16 h before being poured into water (50mL) and extracted with ethyl acetate (3 x 50mL). The combined extracts were dried over magnesium sulfate, filtered and evaporated to give a brown oil which was purified by chromatography on silica gel. Elution with 15 dichloromethane followed by 10% ethyl acetate/dichloromethane gave methyl 3 amino-5-hexamethyleneimino-6-phenyl-2-pyrazinecarboxylate as a yellow solid (0.309 g, 77%). Part 3. NHH 2 0 N HF i. To a solution of sodium (0.090 g, 6.17 mmol) dissolved in methanol (8 nL) was added guanidine hydrochloride (0.598 g, 6.26 mmol) and the mixture was refluxed for 30 min after which it was filtered. To the filtrate was added methyl 3 amino-5-hexamethyleneinino-6-phenyl-2-pyrazinecarboxylate (0.310 g, 0.95 mmol) 25 in tetrahydrofuran (10 mL) and the solution refluxed for 72 h. The solvent was removed under reduced pressure and the residue chromatographed on silica gel.

WO 2004/112687 PCT/AU2004/000866 -58 Blution with 5% methanol/dichloromethane gave N-amidino-3-amino-3 hexamethyleneimino-6-phenyl-2-pyrazinecarboxamide as a yellow solid (0.116 g, 35%). 5 Example 5. Viral Studies Construction of recombinant plasmids containing open reading frames encoding various virus proteins. Complimentary DNA (cDNA) fragments for the various viral proteins listed in Table 2 were obtained either by PCR amplification from a parental virus genome 10 clone, or by direct chemical synthesis of the polynucleotide sequence. For example, the open reading frame encoding Vpu (Fig Ia) was amplified by PCR from a cDNA clone of an Nde I fragment of the IV-1 genome (isolate HXB2, McFarlane Burnet Centre, Melbourne, Australia) as follows: Native Pfu DNA polymerase (Stratagene; 0.035 U//.l) was chosen to catalyse the PCR reaction to minimise possible PCR 15 introduced errors by virtue of the enzyme's proofreading activity. The 5', sense, primer AGTAGGATCCATGCAACCTATACC (<400>2) introduces aBaniHl site (underlined) for cloning in-frame with the 3' end of the GST gene in p2GEX (41). This primer also repairs the start codon (bold T replaces a Q of the vpu gene which is 20 a threonine codon in the HXB2 isolate. The 3', antisense, primer TCTGGAATTLTACAGATCAT CAAC (< 400 >3) introduces an EcoR site. (underlined) to the other end of the PCR product to facilitate cloning. After 30 cycles of 94*C for 45 see, 55*C for 1 min and 72*C for I min in 0.5 ml thin-walled eppendorf tubes in a Perkin-Elmer thermocycler, the 268bp fragment was purified, 25 digested with BamIHi and EcoRI and ligated to p2GEX prepared by digestion with the same two enzymes. The resultant recombinant plasmid is illustrated in Fig lb. The entireVpu open reading frame and the BamHI and EcoRi ligation sites were sequenced by cycle sequencing, using the Applied Biosystems dye-terminator kdt, to confirm the DNA sequence. Other cDNAs were synthesised for us using state of the 30 art methods by GenScript Corporation (New Jersey, USA). Codon sequences were optimised for expression in bacterial, insect or mammalian cells, as appropriate. Restriction endonuclease enzyme recognition sites were incorporated at the 5' and 3' WO 2004/112687 PCT/AU2004/000866 -59 ends of the synthetic cDNAs to facilitate cloning into plasmid expression vectors, pcDNA3.1, pFastBac and pPL451 for expression of the encoded virus proteins in mammalian, insect or bacterial cells, respectively. 5 Standard techniques of molecular biology were used in cloning experiments. For example, to prepare the Vpu open reading frame for insertion into the pPL451 expression plasmid, p2GBXVpu was first digested with BamHI and the 5' base overhang was filled in the Klenow DNA polymerase in the presence of dNTPs. The Vpu-encoding fragment was then liberated by digestion with EcoRl, purified from an 10 agarose gel and ligated into pPL451 which had been digested with Hpal and EcoRI. Western blots subsequently confimed that the pPLVpu construct (Fig Ic) expressed Vpu after induction of cultures at 42 0 C to inactivate the c1857 repressor of the PR and PL promoters. Table 2 Source of viral eDNA or peptide sequences. Target Protein Source organism Strain or Sequence Accession number Vpu HIV-1 strain HXB2 SARS-CoV E protein SARS coronavinis P59637 HCV p7 Hepatitus C virus H77 la NP 751922 MHV-E protein Murine hepatitis virus NP 068673 229E E protein Human coronavirus 229E NP 073554 Dengue M protein Dengue virus type 1 Strain Singapore S275/90 Example 6. Purification of Recombinant Vpu from E. Colt Cultures of E. coff strain XLI-blue cells containing p2GEXVpu were grown at 30*C with vigorous aeration in LB medium supplemented with glucose (6g/L) and 20 ampicillin (50mg/L) to a density of approximately 250 Klett units, at which time IPTG was added to a final concentration of 0.01mM and growth was continued for a further 4hr. The final culture density was approximately 280 Klett units. Since early experiments revealed that the majority of expressed CST-Vpu fusion protein was associated with both the cell debris and 30 membrane fractions, the method of 25 Varadhachary and Maloney (Varadhachary and Maloney, 1990) was adopted to isolate osmotically disrupted cell ghosts (combining both cell debris and membrane fractions) for the initial purification steps. Cells were harvested, washed, weighed and WO 2004/112687 PCT/AU2004/000866 -60 resuspended to 10ml/g wet weight in MTPBS containing DTT (ImM) and MgC12 (10mM). Lysozyme (0.3 mg/ml; chicken egg white; Sigma) was added and incubated on ice for 30 min with gentle agitation followed by 5 min at 37 0 C. The osmotically sensitised cells were pelleted at 12,000g and resuspended to the origial volume in 5 water to burst the cells. The suspension was then made up to IxMTPBS/DTT using a l0x buffer stock and the ghosts were isolated by centrifugation and resuspended in MTPBS/DTT to which was then sequentially added glycerol (to 20 % wt/vol) and CHAPS (to 2 % wt/vol) to give a final volume of one quarter the original volume. This mixture was stirred on ice for 1 hr and then centrifuged at 400,000g for 1hr to 10 remove insoluble material. The GST-Vpu fusion protein was purified from the detergent extract by affinity chromatography on a glutathione agarose resin (Sigma). The resin was thoroughly washed in 50mM Tris pH 7.5 containing glycerol (5 %), DTT (mM), and CHAPS (0.5 %) (Buffer A) and then the Vpu portion of the fusion protein was liberated and eluted from the resin-bound GST by treatment of a 50% 15 (v/v) suspension of the beads with human thrombin (100U/ml; 37*C for lhr). PMSF (0.5mM) was added to the eluant to eliminate any remaining thrombin activity. This Vpu fraction was further purified on a column of MA7Q anion exchange resin attached to a BioRad EPLC and eluted with a linear NaC1 gradient (0-2M) in buffer A. 20 The Vpu was purified to homogeneity - as determined on silver stained gels - on an immunoaffinity column as follows: HPLC fractions containing Vpu were desalted on a NAP 25 column (Phanracia) into buffer A and then mixed with the antibody agarose beads for 1hr at room temperature. The beads were washed thoroughly and Vpu was eluted by increasing the salt concentration to 2M. Protein was quantitated 25 using the BioRad dye binding assay. Example 7. Expression and Purification of VDu in E.Colh The plasmid p2GEXVpu (Fig. 1) was constructed to create an in-frame gene fusion between the OST and Vpu open-reading frames. This system enabled ITG-inducible 30 expression of the Vpu polypeptide fused to the C-terminus of GST and allowed purification of the fusion protein by affinity chromatography on glutathione agarose..

WO 2004/112687 PCT/AU2004/000866 -61 Optimal levels of GST-Vpu expression were obtained by growing the cultures at 30"C to a cell density of approximately 250-300 Klett units and inducing with low levels of IPTG (0.01mM). To purify the GST-Vpu, a combined cellular fraction containing the cell debris and plasma membrane was prepared by lysozyme treatment 5 of the induced cells followed by a low-speed centrifugation. Approximately 50% of the GST-Vpu protein could be solubilised from this fraction using the zwitterionic detergent CHAPS. Affinity chromatography using glutathione-agarose beads was used to enrich the fusion protein and-thrombin was used to cleave the fusion protein at the high affinity thrombin site between the fusion partners, liberating Vpu (Fig. 10 2A). In fractions elated from the anion exchange column Vpu was the major protein visible on silver stained gels (Fig. 23, lane 1). Finally, Vpu was purified to apparent homogeneity on an immunoaffinity column (Fig. 2B, lane 2). The N-terminal amino acid sequence of the protein band (excised from SDS-PAGB gels) corresponding to the immunodetected protein confirmed its identity as Vpu. 15 Example 8. Reconstitution of Vipu in Phospholipid Vesicles. Proteoliposomes containing Vpu were prepared by the detergent dilution method (New, 1990). A mixture of lipids (PB:PC:PS; 5:3:2; hng total lipid) dissolved in cbloroforn was dried under a stream of nitrogen gas and resuspended in 0.1 ml of 20 potassium phosphate buffer (50mM pH 7,4) containing DTT (ImM). A 25pl aliquot containing purified Vpu was added, followed by octylglucoside to a final concentration of 1. 25 % (wt/vol). This mixture was subject to three rounds of freezing in liquid nitrogen, thawing and sonication in a bath type sonicator (20-30 see) and was then rapidly diluted into 200 volumes of the potassium phosphate buffer. 25 Proteoliposomes were collected by centrifugation at 400,000g for lbr and resuspended in approximately 150pl of phosphate buffer. Example 9. Assaving Vp Ion Channel Activity Purified Vpu was tested for its ability to induce channel activity in planar ipid bilayers using standard techniques as described elsewhere (Miller, 1986; and Piller et 30 al, 1996). The solutions in the CIS and TRANS chambers were separated by a DehinTm plastic wall containing a small circular hole of approximately 100pm WO 2004/112687 PCT/AU2004/000866 -62 diameter across which a lipid bilayer was painted so as to form a high resistance electrical seal. Bilayers were painted from a mixture (8:2) of pahnitoyl-oleoly phosphatidyl-ethanolamine and pahnitoyl-oleolyphosphatidyl-choline (Avanti Polar Lipids, Alabaster, Alabama) in n-decane. The solutions in the two chambers 5 contained MES buffer (10mM, pH 6.0) to which various NaCI or KC1 concentrations were added. Currents were recorded with an AxopatchTM 200 amplifier. The electrical potential between the two chambers could be manipulated between +/-200mV (TRANS relative to grounded CIS). Aliquots containing Vpu were added to the CIS chamber either as a detergent solution or after incorporation ofthe protein into 10 phospholipid vesicles. The chamber was stirred until currents were observed. Examole 10. Vpu Forms Ion Channels in Lipid Bilayers. To assay for ion-channel formation by Vpu, reconstitution into planar lipid bilayers was perfbnned. When samples (containing between 7 and 70ng of protein) of purified recombinant Vpu were added to the iml of buffer in the CIS chamber of the 15 bilayer apparatus, current fluctuations were detected after periods of stirring that varied from 2 to 30 min (Fig. 3). This time taken to observe channel activity approximately correlated with the amount of protein added to the chamber. No channels were detected when control buffer aliquots or control lipid vesicles were added to the CIS chamber. In those control experiments the chambers could be stirred 20. for more than an hour without appearance of channel activity. Example 114 Properties of The Vu Channels. Channel activity was observed in over 40 individual experiments with Vpu samples prepared from five independent purifications. In different experiments, the amplitude of the currents varied over a large range and, again, seemed to 25 approximately correlate with the amount of protein added. The smallest and largest channels measured had conductances of 14 pS and 280 pS, respectively. The channels were consistently snaller when lipid vesicles containing Vpu were prepared and fused to the bilayer rater than when purified protein in detergent solution was added. This may be because the former method included treatment with high concentrations 30 of detergent and a dilution step that may have favoured the breakdown of large aggregates into monomers.

WO 2004/112687 PCT/AU2004/000866 -63 The relationship between current amplitude and voltage was linear and the reversal potential in solutions containing a ten-fold gradient of NaC1 (500mM CIS; 50mM TRANS) was +3OmV (Fig. 3B). A similar reversal potential was obtained when solutions contained KCI instead of NaCl. In 5 experiments with either NaC1 or 5 KCI in the solutions on either side of the membrane, the average reversal potential was 31.0 +/-1.2mV (+/-SEM). This is more negative than expected for a channel selectively permeable for the cations alone. Using ion activities in the Goldman Hodgkin-Katz equation gives a P/Pj ratio of about 5.5 indicating that the channels are also permeable to chloride ions. An attempt was made to reduce the anion current 10 by substituting phosphate for chloride ions. When a Na-phosphate gradient (150mM Na* & 100mM phosphate CIS; 15mM Na & 10mM phosphate TRANS, pH 6.8) was used instead of the NaCl gradient, the reversal potential was 37.1 +/- 0.2 (+/-SEM, n=2) again indicating a oation/anion permeability.ratio of about 5. (For calculations involving the phosphate solutions, the summed activities of the mono and bivalent 15 anions were used and it was assumed that the two species were equally permeable). The current-voltage curve now exhibited rectification that was not seen in the NaC1 solutions. It can be concluded that the channels formed by Vpu are equally permeably to Na and K+ and are also permeable, though to a lesser extent, to chloride as well as phosphate ions, 20 Example 12. Bacterial Bio-Assav for Screening Potential Ion Channel-Blocking Drus.. This bio-assay is based on the observation that expression of Vpu in K col results in an active Vpu channel located in the plasmalemma that dissipates the transmembrane sodium gradient. As a consequence of this Vpu channel activity, 25 metabolites whose accumulation within the colls is mediated by a sodium dependent co-transporter (for example proline or adenine) leak out of the cell faster than they can'be synthesised so that the metabolites' intracellular levels become limiting for growth of the cell. Thereby, an E. colt cell expressing Vpu is unable to grow in minimal drop-out media lacking adenine or proline. However, in the presence of a 30 drug that blocks the Vpu channel, the cell is once again able to re-establish its transmembrane sodium gradient - due to the action of other ion pumps in the membrane - and the leakage of metabolites is prevented enabling growth.

WO 2004/112687 PCT/AU2004/000866 -64 Experiments to demonstrate that Vpu can form sodium channels in the plasma membrane ofE. coil were performed as follows. To express infused Vpu in E. coli, the vpu open-reading frame was cloned into the plasmid pPL45,1 to create the recombinant plasmid pPL-Vpu (Fig. lb). In this 5 vector the strong PL and PR lambda promoters are used to drive expression of Vpu under control of the temperature sensitive c1857 represser, such that when grown at 30"C expression is tightly repressed and can be induced by raising the temperature to between 37*C and 42*C. On agar plates, cells containing pPL-Vpu grew when incubated at 30*C and 37*C but not at 42*C, while control strains grew well at 42C. 10 Liquid cultures of cells containing pPL-Vpu were grown at 30*C to 0Doo,=0.84 then moved to grow at 42"C for two hours (the final cell density was OD 6 oo,=0.75). The plasma membrane fraction was prepared and western blotting, using an antibody that specifically binds to the C-terinus of Vpu, detected a single band at approximately l6kDa, indicating that Vpu was expressed and associated with the membranes (Fig. 1$ 2A, lane 5). Example 13. Cross-Feedine Experiments Reveal That Proline Leaks Out of Cells Expressing Vu. Uptake of proline by E. colt is well characterised and active transport of the amino acid into the cells is known to use the sodium gradient as the energy source 20 (Yamato et al, 1994). To detect whether praline leakage occurs, the following cross feeing assay was used: A lawn of an E. coli strain auxotrophic for proline and methionine (Met' Pro), was seeded and poured as a soft agar overlay on minimal drop-out media plates lacking proline but containing metbionine. Sterile porous filter discs were inoculated with a Met Pro' stain (XL-1 blue) containing either the 25 pPL451 control plasmid or pPL-Vpu and placed onto the soft agar. The plates were then incubated at 37*C or 30 0 C for two days. After than time a halo growth of the Mef Pro' strain was clearly visible surrounding the disc inoculated with the cells. containing pPL-Vpu incubated at 37*C (Fig. 4A). This growth can only be due to the leakage of praline from the Vpu-expressing cells on the disc. No such leakage was 30 apparent from the control strain at 37*C nor around either strain on plates grown at 30*C (Fig. 4B).

WO 2004/112687 PCT/AU2004/000866 -65 In contrast to proline transport the E. coli methionine permease is known to belong to the ABC transporter family (Rosen, 1987) and hence be energised by ATP. Identical crossfeeding experiments to those described above were set us except that the Mef Pro strain was spread on minimal drop-out plates lacking methionine but 5 containing proline. No growth of this strain was evident around any of the discs (Fig. 4C), indicating that methionine was not leaking out of the XL-1 blue cells even when Vpu was being expressed. Example 14. E.Coli Cells Expressing Vm Require Adenine in the External Medium for Growth. 10 It was observed that, due to an uncharacterised mutation in the adenine synthesis pathway, growth ofE. colt cells of the XL-blue strain expressing Vpu at 37*C was dependent on the presence of adenine in the medium. This allowed the development of an even simpler bioassay for Vpu ion-channel activity than the proline cross-feeding assay described above: A lawn of XL1-blue cells containing the 15 pPL-Vpu plasmid is seeded onto an agarose plate lacking adenine in the medium, small aliquots of drugs to be tested for inhibition of the Vpu channel are spotted onto the agarose in discrete locations and the plates are incubated at 37*C for a suitable period of time (12-36 hours). Halos of growth around a particular drug application site indicate that the drug has inhibited expression of the Vpu ion channel activity that 20 prevents growth in the absence of the drug. (Figure 5). Example 15 Assay of Compounds in Planar Livid Bilavers for Vpu Channel Blocidng. Activity 25 Comopunds were characterized for their ability to block Vpu ion channel activity reconstituted into planar lipid bilayers. Vpu N-terminal peptide (residues 1 32) dissolved in trifluoroethanol was added to the CIS chamber of the bilayer apparatus and the solutions was stirred until ion currents were observed, indicating incorporation of one or more Vpu ion channels into the bilayer. After recording the 30 channel activity for a few minutes, drugs were added to the solutions in the CIS and TRANS chambers - with stirring - to a final concentration of lOOMM. Channel activity was then recorded for at least a further three minutes and the effect of drug addition on ion current was determined by comparing the channel activity before and WO 2004/112687 PCT/AU2004/000866 -66 after drug addition. For each experiment, drug effect was classified into four categories: "Stong block", if current was inhibited approximately 90-100%; "weak block", approx. 50-90% inhibition; "partial block", <50%; and "no effect". Experiments were disregarded if currents larger than ±50pA were generated after 5 addition of Vpu N-peptide because in such cases it is possible that non-native peptide aggregates contribute to bilayer breakdown. Such aggregates, by virtue of their disorganized structure may not be specifically blocked by the drugs at the concentrations tested. 10 Table 3 summarises the results of the bilayer experiments. A novel outcome of these experiments was the strong blocking of Vpu channels observed with Phenamil. Phenamil has a phenyl group derivative at the guanidine group of amiloride, Amiloride itself is not a blocker of Vpu, whereas addition of the hexamethylene group at the 5- position of the pyrazine ring created a structure (HMA) that blocks the 15 channel at concentrations as low as 2SpM. These new results with Phenamil, however, now show that a bulky hydrophobic derivative at the opposite end of the molecule can also turn amiloride into an effective Vpu channel blocker. Interestingly, benzamil, with a very similar structure was much less effective at blocking the Vpu channel. 20 Table 3: Summary of Compounds Inhibiting the Vpu Ion Channel in Bilayers Compound No. of Results Expts. Phenamil .3 3x Strong block MIA 2 Ix Strong block; 1x weak Benzamil 10 3x partial block; 7x no effect BIPA 3 3x weak block; HIMA I x Strong block; (5-Phenyl-penta-2,4-dienyl)guanidine 6 6x strong block 6-methoxy-2-naphthoy1guanidine . 5 5x strong block (2-Chlorocinnamoyl)guanidine 6 4x strong; 2x partial blocks 3-(trifluoromethyl)cinnamoylguanidine 5 4x strong blocks; 1x no effect WO 2004/112687 PCT/AU2004/000866 -67 N-{S-[3-(5-Guanidino-pentyloxymethyl). benzyloxy]-pentyl)-guanidine 4 3x strong block; 1x no effect 4-phenylbenzoylguanidine 3 3x strong block 3-methyloinnamoylguanidine 4 2x strong block; 2x partial (3-Chlorocinnamoyl)guanidine 4 2x strong block; 2x partial N-(3-phenylpropanoyl)-N' phenylguanidive I lx strong blocks (3-Bromocinnamoyl)guanidine 3 3x partial-strong block 5-tert-butylamino-amiloride 3 3x partial block N-amidino-3-amino-5-phenyl-6-chloro-2 pyrazinecarboxamide 3 3x partial block 3-methoxy -HMA 3 3x partial block 5-(N-Methyl-N-isobutyl)amniloride 1 1x partial block 5-(N-Ethyl-N-isopropyl)amiloride 1 1x partial block 2-napthoylguanidine 7 7x weak block N,N'-bis(3phenylpropanoy1)-N" phenylguanidine 7 7x weak block cinnamnoylguanidine 3 3x weak block (5-Phenyl-penta-2,4-dienoyluanidine 6 6x strong block Example 16 Compound Screening using the Bacterial Bio-Assay for the Vou protein. 5 The halos of growth around the site of application of particular drugs - as described in example 14- were given a score between zero and six reflecting the size and density of the zone of bacterial cell growth. Scores greater than 3 represent strong inhibition of the Vpu protein; scores between 1.5 and 3 represent moderate inhibition and scores between 0.01 and 1.5 represent fair inhibition. 10 Table 4 lists the scores for inhibition of Vpu protein in the bacterial bio-assay. Table 4 Vpu Inhibition (score /# of times Compound tested) (3-Chlorocinmamoy)guanidine 4.38/4 3-Bromocinamoyl)guanidine 4.3/24 2-Chlorocinnamoy)guanidine 4.0/4 2-Bromocinnamoyl)guanidine 3.7/2 -(trifluoromethy1)cinnamoylguanidine 3.7/2 WO 2004/112687 PCT/AU2004/000866 -68 5-bromo-2-fluorocinnamoylguanidine 3.5/2 3-metbylcinnamoylguanidine 3.4/2 2-methylcinnamoylguanidine 3.1/2 2,3-dimethylcinnmoylguanidine 3.1/2 innaimoylguanidine 2.96/12 6-methoxy-2-naphthoylguanidine 2.9/4 trans-3-(1-napthyl)acryloyguanidine 2.9/3 3,4-dichlorocinnamoylguanidine 2.9/3 2,6-dichilorocinnamoylguanidine 2.88/2 -phenylbenzoylguanidine 2.75/5 -ethyleinnamoylguanidine 2.75/2 (4-Chlorocinnamoyl)guanidine 2.7/5 2-naptboylguanidine 2.7/11 ,5-dimethyleinnamoylguanidine 2.69/2 -isopropylcinnamoylguanidine hydrochloride 2.6/2 5-Phenyl-penta-2,4-dienyl)guanidine -2.56/2 -phenylominnmoylguanidine 2.54/3 (4-Bromocinnamoyl)guanidine 2.5/4 5-(3'-bromophenyl)penta-2;4-dienoylguanidine 2.5/2 3-(cyclohex-1-en-1-yl)einnamoylguanidine 2.5/2 3-(trifluoromethoxy)cinnamoylguanidine 2.44/2 2-(tdfluorometlhyl)cinnamoyguanidine 2.4/2 N,N-bis(3phenylpropanoy1)-N"-phenylguanidine 2.25/3 2-ethoxycinnmoylguanidine 2.25/2 N-(3-phenylpropanoyl)-N'-phenylguanidine 2.21/3 4-(trifluoromethyl)cinnamoylguanidine 2.2/2 (4-Methoxycinnamoyl)guanidine 2,13/3 2-t-butycinamoylganidine 2.13/2 4-methylinnamoylguanidine 2.1/2 2-fluorocinnamoylguanidine 2.1/2 2-phenycinnamoylguanidine 2.1/2 N-(6-Hydroxy-2-napthoy)-N'-phenylguanidine 2.06/2 3-t-butyleinnamoylguanidine 2.06/2 3,4-difluorocinanioylguanidine 2.06/2 5-(NN-hexamethylene)amiloride 1.9/31 3-fluorocinmaoylguanidine 1.9/2 5-bromo-2-methoxycinnamoylguanidine 1.9/2 3-ethoxycinnamoylguanidine 1.9/2 3,4-(methylenedioxy)cinnamoylguanidine 1.88/2 (2-Methoxyoinnamoyl)guanidine 1.7/4 '4 DichloroBenzamil HCI 1.7/2 2,3,5,6,-tetramethyleinnamoylguanidine 1.6/2 -(2-napthyl)acryloylguanidine 1.56/2 2-(1-napthyl)acetoylgunidine 1.56/2 2,3-difluorocinanaoylguanidine 1.56/2 (3-Methoxycinnamoyl)guanidine 1.52/6 4-isopropyleimnamoylguanidine 1.4/2 2,4,6-trimethyleinnamoyguanidine 1.4/2 WO 2004/112687 PCT/AU2004/000866 -69 N-(cinamoy1)-N'phenylguaidine 1.25/3 2-(cyclohex-1 -en-lyl)cinnamoylguanidine 1.21/2 2-(2-napthyl)acetoylguanidine 1.19/2 (4-Hydroxycinnamoyl)guaidine 1.1/2 -phenylcinnamoylguanidine 1.1/2 4-fluorocinnamoylguanidine

.

1.1/2 ,N'-bis-(cinnamoyl)-N"-phenylguanidine 0.94/2 (2-Furanacryloyl)guanidine 0.94/2 Phenamil metbanesulfonate salt 0.9/5 Benzamil hydrochloride 0.9/3 (3-Nitrocinnamoy)guanidine 0.9/1 Benzyoylguanidine 0,88/2 (4-henoxybenzoy)guanidine 0.81/2 3-(trans-hept-1-en-1-y1)cinnamoylguanidine 0.81/2 5-(N-Methyl-N-isobutyl)aiiloride 0.8/2 2-cyclohexylcinnamoylguanidine 0.8/2 4-ethoxycinnamoylguanidine 0.69/2 2,4-dichlorocinnamolyguanidine 0.63/2 5-(N-Ethyl-N-isopropyl)amiloride 0.6/3 N-amidino-3-aino5-hexmethyleneimino-6-phenyl -pyrazinecarboxamide 0.6/2 (a-Methylcinnamoyl)guanidine 0.6/2 Gimeoylguanidine hydrochloride 0.6/2 [(4-Chlorophenoxy-aety]guanidine 0.56/2 N-amidino-3-amino-5-phenyl-6-chlero- 2 pyrazinecarboxamide 0.5111 S-(4-fluorophenyl)amiloride 0.4/6 (trans-2-Pheny1cyclopropanecarbony1)guanidine 0.4/2 (2-Nitrocinnamoyl)guanidine 0.4/2 trans-3-Furanacryoylguanidine 0.38/2 1-napthoylguanidine 0.3/2 -tert-butyamino-amiloride 0.217 3-methoxy -HMA 0.2/4 (3-phenylpropanoyl)guanidine 0.2/4 4-t-butyleinnamoylguanidine 0.19/2 5-(N,N-Dimethy1)amiloride hydrochloride 0.1/2 ,N'-Bis(3-phenylpropanoyl)guanidine 0.1/2 N-Benzoyl-N-cinnamoylguanidine 0.06/2 1-bromo-2-napthoylguanidine 0.06/2 Example 17. Effect of Compounds on HIV Relication in Human Monocytes and Macrophages. Human monocytes were isolated from peripheral blood and cultured either for 5 24hr (one day old monocytes) or for 7 days to allow differentiation into monocyte derived macophages (MDM). These cells were then exposed to cell-free preparations WO 2004/112687 PCT/AU2004/000866 -70 of HV isolates and allowed to absorb for 2hr before complete aspiration of the medium, washing once with virus-free medium and resuspension in fresh medium. The cells were exposed to various concentration of compound either 24 hr prior to infection or after infection. Subsequent HIV replication, at various times after 5 infection, was compared in cells exposed to drugs and in cells not exposed to drugs (controls). The progression and extent of viral replication was assayed using either an HIV DNA PCR method (Fear et al, 1998) or an ELISA method to quantitate p 2 4 in culture supernatants (Kelly et al, 1998). Table 5 provides examples of results obtained using this assay and test 10 antiviral compounds. Table 5 Drug Pereent of Cone. Positive Compound pM Control None - positive control 100% 4-phenylbenzoylguanidine 10 26 5 4. 2.5 9 1. 216 0.625 10 None - positive control 100% (3-Bromocinnamoyl)guanidine 10 3 5 1 2.5 9 1.25 59 0.625 116 None - positive control . 100% 3-(trifluoro- 10 11 methyl)cinnamoylguanidine 5 8 2.5 25 1.25 27 0.625 38 None -positive control 100% 10 6 hexamethylene)amiloride 5 21 2.5 50 1.25 19 0.625 30 WO 2004/112687 PCT/AU2004/000866 -71 Example 18. SARS Coronavirus. SARS E rotein forms an ion channel Peptide Synthesis A peptide corresponding to the full-length SARS-CoV (isolate Tor2 and - 5 Urbani) E protein (MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCA YCCNIVNVSLVKPTVYVYSRVKNLNSSEGVPDLLV) and a second peptide comprising the first 40 amino acids of the full length E protein which correspond to the transmembrane domain (MYSFVSEETGTLIVNSVLLFLAFVVF LLVTLAILTALRLC) were synthesized manually using FMOC chemistry and solid 10 phase peptide synthesis The synthesis was done at the Biomolecular Resource Facility (John Curtin School of Medical Research, ANT, Australia) using a Symphony Peptide Synthesiser from Protein Technologies Inc.(Tucson, AZ, USA) according to the manufacturers instructions. Example 19. Peptide purification 15 Mass spectral analysis of the synthetic peptide revealed that the preparation contained significant amounts of material with lower m/z ratio than expected for the full-length product. The majority of these are presumably tnmcated peptides generated during the peptide synthesis process. To enrich the full-length E protein, the following procedure was used, which relies on differential solubility of the 20 smaller molecules and full-length peptide. The crude preparation was suspended at 12 mg/mil in 70% CH 3 CN, 0.1 %TFA and vortexed for 10 minutes. This suspension was centrifuged at 10,000g for 10 minutes at 20"C, The supemataot was discarded and the insoluble fractions was extracted with 70% CH 3 CN, 0.1% TFA, as above, two more times. The insoluble material containing the E protein was dried using 25 Speedvac an the weight of the final product was used to calculate the yield. The purified peptide was analysed by Bruker Omniflex MALDI-TOF mass spectrometry in HABA matrix at 2.5mg/mi in methanol at a 1:1 ratio and spectra were obtained in the positive linear mode. A clear peat at m/z ratio of 8,360.1 was seen as expected for the calculated molecular weight of full-length E protein and 4422.3 for the N-terminal 30 E protein.

WO 2004/112687 PCT/AU2004/000866 72 Example 20. Planar Linid Bilavers The SARS vius B protein was resuspended at 1mg/ml in 2 ,2,2-trifluoroethanol. The SARS virus E protein's ability to form ion channels was tested on a Warner (Warner instruments, Inc. 1125 Dixwell Avenue, Ilamden, CT 06514) bilayer rig as follows; A 5 lipid mix of 3:1:1, 1-Palmitoyl-2-oleolyl phosphatidyl Ethanolamine: 1 -Pahnitoyl-2 oleolyl phosphatidyl Serine: 1-Palmitoyl-2-oleolyl phosphatidyl choline in CHC was dried under N 2 gas and resuspended to 50mg/ml in n-decane, Bilayers were painted across a circular hole of approximately 100pm diameter in a Delrin cup separating aqueous solution in the CIS and TRANS chambers. The CIS chamber 10 contained a solution of 500mM NaCi or KCI, in a 5mM HEPES buffer pH 7.2, the TRANS chamber contained a solution of 50mM NaCl or KC, in a 5mM HEPES buffer pH 7.2. Silver electrodes coated in chloride with 2% agarose bridges are placed in the CIS and TRANS chamber solutions. The SARS B protein full-length or N tenninal peptides (31 Ong) were added to the CIS chamber, which was stirred until 15 channel activity was detected. The CIS chamber was earthed and the TRANS chamber was held at various holding potentials ranging between +100 to -100mV. Currents were recorded using a Warner model BD-525D -amplifier, filtered at 1kHz, sampling at 5 kHz and digitally recorded on the hard disk of a PC using software developed in house. 20 Drugs to be tested for their ability to inhibit SARS E protein ion channel activity were made up at 50mM in a solution of 50% DMSO: 50% methanol. For experiments testing the ability of compounds to inhibit B protein ion channel activity, 100 M to 400 pM of compound was added to the CIS chamber while stirring for 30 seconds. Bilayer currents were recorded before channel activity, during channel 25 activity and after the addition of the drug, Among the compounds tested was cinnamoylguanidine (BitO36), a compound which was shown in earlier experiments to be antiviral and to inhibit ion channel proteins from other viruses. Example 20.1. Polyaerylamide gel electrophoresis 30 Purified E protein was dissolved to 1 mg/ml, 5 mg/ml and 10 mg/ml in, 6 M Urea, 10% Glycerol, 5% SDS, 500 mM DTT, 0.002% Bromophenol Blue, 62.5 mM Tris HC1 (pH 8.3). Peptides in solutions were heated at IOOC for 20 minutes before WO 2004/112687 PCT/AU2004/000866 -73 30 pL samples were run on stacking gel 4-20% (Gradipore). SeeBlueOpre-stained standard (Invitrogen) was used for molecular weight markers. Example 20.2 Results To test if the SARS E protein forms ion channels the purified synthetic 5 peptide was reconstituted into planar lipid bilayers (21). Typically, 3pg of SARS fhll-length E protein was added to the CIS chamber, while stirring. This CIS chamber contained 500 mM NaCl and the TRANS chamber contained 50 mM NaCl. In 60 experiments, ion currents due to SARS E protein ion.channel activity were observed after about 5 -15 minutes of stirring. Activity was detected more rapidly and reliably 10 with a holding potential of approximately-100mV across the bilayer. Currents recorded at -100mV, (A) and at -60mV (B) in one of these experiments are shown in Figure 6. In that experiment the reversal potential was about +48mV and the channel conductances were calculated to be 52pS and 26pS, respectively. This indicates that the curent-voltage (IV) relationship is not linear. In ten other experiments, where no 15 protein was added to the CIS chamber, no ion channel activity was detected, even after recording for over 1 hour. Figure 7a. shows typical current traces recorded ever a range of potentials in NaC1 solutions. In that experiment the direction of current flow reversed at +48mV (Fig 7b). The 1V curve shows that at the lower voltages the average current flow across the 20 bilayer is small but at higher potentials there is an increase in average current across the bilayer, resulting in a non-linear IV relationship, In seven independent experiments, the average reversal potential was +48.3 ± 2.3 mV (mean ± 1SEM), indicating that the chanthels were about 37 times more permeable to Na+ ion than to Ca ions. The reversal potential is close to the Na+ equilibrium potential (+53mV), 25 therefore the channel is selective for Na+ ions. For these 7 experiments the channel conductance varied between 95-164 pS; the average conductance was 130 ±13 pS. SARS E protein ion channel is slightly less selectivity for K ions than Na* ions. Figure 8b shows recording of currents in KCI solutions at a range of potentials. In this experiment the currents reversed at +31 mV. In seven similar experiments E 30 protein ion channel average reversal potential was +34.5 t 2.5 mV. Therefore the SARS E protein ion channel is about 7.2 times more permeable to KCions than Cr WO 2004/112687 PCT/AU2004/000866 -74 ions. In seven experiments, the channel conductance varied ranging between 24-166 pS, the average conductance was 83.4 ±26 pS. Similar results were obtained with a second synthetic peptide, which corresponded to the first forty N-terminal amino acids of the SARS E protein "N-terminal peptide" 5 (21). The average reversal potential in NaCI solution in four experiments was +46.3 2.5 mV, indicating that the ion channel formed by N-terminal peptide is about 25 times more permeable to Na+ ion than to Cl-ions. The SARS E protein N-terminal peptide was sufficient for the formation of ion channels with properties like those of the full length SARS E protein. Therefore, the selectivity filter for the SARS E 10 protein is most likely contained within the first forty amino acids of the N-terminal. SARS E protein N-terminal peptide also formed ion channels in KC1 solution that were similarly selective for K+ ions compared to the full-length E protein. In five independent experiments the average channel reversal potential was +39.5 ± 3.6 mV, therefore the channel is about 11 times more permeable to K' ions than Cl ions. 15 SDS-PAGE of the purified full-length E protein peptide showed bands corresponding to the full-length E protein (Data not shown). Larger bands of varying size up to about 20 kDa were detected, suggesting that SARS E protein may form homo oligomers. 20 Example 21. SARS E protein ion channel is blocked by cinnamovlguanidine and other compounds E protein ion channel activity in NaCl solutions was significantly reduced (p 0.01, n=6 experiments) by addition of 100 to 200 pM cinnamoylguanidine to the CIS chamber. The average current across the bilayer was reduced to baseline by 25 100pM cinnamoylguanidine. In experiments when E protein ion channels had higher conductance, 100 to 200 pM cinnamoulguanidine reduced the average current across the bilayer about 4 fold. Similarly, in four other experiments, 100 to 200 pM cinnamoylguanidine blocked channels formed by full-length E protein in KCI solutions. In two additional experiments, the SARS E protein N-terminal peptide was 30 blocked by 100 to 200 pM cimnamoylguanidine, demonstrating that the cinnamoylguanidine drug-binding site is located within the first forty amino acids of WO 2004/112687 PCT/AU2004/000866 -75 the B protein N-terminal domain. Other compounds tested in bilayers for their effect on the SARS E protein are shown ii below in Table 6. Table 6 5 % Reduction of ave Co~npournd current by 100 5-(N,N-hexamethylene)amiloride 91 ±7 6-methoxy-2-naphthoylguanidine 92 ±16 2'4 DichloroBenzamil HC1 78± 0 N,N'-bis(3phenylpropanoy)-N"-phnylgpanidne 88 ± 6 (3-Bromocinnamoyl)guanidine 87± 11 (2-Bromocinnmnoyl)guanidine 88± 6 trans-3-(1-napthyl)acryloylguanidine 66± 2 Examle 21.1 Results and Discussion. We have shown that SARS E protein can fonn ion channels in lipid bilayer 10 membranes. The ion currents reversed at positive potentials, which demonstrates that E prtein ion channels are selective for monovalent cations over monovalent anions. E protein Ion channels were about 37 times more selective for Na+ ions over Cl-ions and about 7.2 times more selective for K+ ions over Cl- ions. In over 60 experiments the Na+ conductance of the E protein ion channel varied from as low as 26 pS to as 15 high as 164 pS. SDS-PAGE showed that the E protein forms homo-oligomers, and we sunmised that the larger conductances were probably due to aggregation of the E protein peptide leading to larger ion channels or the synchronous opening of many ion channels. Single channel currents were observed in several experiments and from these the chmmel conductance was calculated to be voltage dependent. 20 The first 40 amino acids of the N-terminal which contains the hydrophobic domain of the SARS virus E protein is sufficient for the formation of ion channels on planar lipid bilayers. The N-terminal E protein ion channel has the same selectivity and conductance as the full-length E protein ion channel. The SARS virus fnll length E protein ion channel activity and N-terminal 25 domain E protein ion channel activity on planar lipid bilayers in NaC1 and KC1 solutions was inhibited by addition of between 100pM to 200pM cinnamoylguanidine to the CIS chamber. Inhibition or partial. inhibition of the E WO 2004/112687 PCT/AU2004/000866 -76 protein ion channel activity by cinnamoylguanidine has been observed in seven independent experiments in NaCJ solution and four independent experiments in KCI solution. All known coronaviruses encode an E protein with a hydrophobic N-terminus 5 transmembrane domain therefore all coronaviruses E proteins could form ion channels on planar lipid bilayers. This indicates that the E protein could be a suitable target for antiviral drugs and potentially stop the spread of coronavirus from infected host cells. Drugs that block the E protein ion channel could be effective antiviral therapy for the treatment of several significant human and veterinary coronavirus 10 diseases including SARS and the common cold. Example 22. Bacterial Bio-Assay for Screening Potential SARS-CoV Eproteln Ion Channel-Blocking Drugs. 15 SARS-CoVE protein Ion Channel inhibits Bacterial Cell growth. A bio-assay of SARS-CoV E protein fiction in bacterial cells was developed. A synthetic cDNA fragment encoding SARS-CoV E protein was cloned into the expression plasmid pPL451, creating a vector in which E protein expression is temperature inducible, as described in Example 4. Inhibition of the growth of E.coli 20 cells expressing E protein at 37"C was observed as an indicator of p7 ion channel function dissipating the normal Na+ gradient maintained by the bacterial cells. Example 23. Compound Screening usina the Bacterial Bio-Assay for SARS coronavirus E protein. 25 The halos of growth around the site of application of particular drugs - as described in example 14 -were scored as decribed in example 15. Table 7 lists the scores for inhibition of SARS-CoV E protein in the bacterial bio 30 assay. Table 7 SARS E protein Inhibition (score /# of times Compound tested) WO 2004/112687 PCT/AU2004/000866 -77 ,3-difluorocinnamoylguanidine 4.5011 ,4-dichlorocinnamoylguanidine 4.15 12 4-t-butylcinnamoylguanidine 4.00/1 3-(2-napthy1)acryloylguanidine 3.88/ 1 (3-Chlorooinnamoy1)guanidine 3.87/3 -(cyclohex-1-en-1-yl)cinnamoylguanidine 3.75/1 25-dimethyloinnamoy1guanidine 3.63/1 ans-3-(1-napthyl)acryloylguanidine 3.38 /2 -isopropylcinamoylguanidine 3.16/2 3-Bromocinnamoyl)guanidine 3.15/27 -methoxy-2-naphthoylguanidine 3.13/3 -(N-Methyl-N-isobutyl)amiloride 3.13/2 3-phenylcinnamoylguanidine 3.13/1 (2-Chlorocinnamoyl)guanidine 3.1/3 2'4 DichloroBenzamil HC1 3.00/2 4-phenylcinnamoylguanidine 2.75 /2 4-(trifluoromethyl)cinnamoylguanidine 2.75/1 3-(trifluoromethoxy)einnamoylguanidine 2.71 /1 3-(trifluoromethyl)cinnamoylguanidine 2.67/1 2-ethoxycinnamoylguanidine 2.57/1 cinnamoylguanidine hydrochloride 2.50/1 4-ethoxycinnamoylguanidine 2A8 /2 (2-Bromocinnamoylguanidine 2.47/3 2,6-dichlorocinnamoylguanidine 2.25 /1 ,4,5-trimethoxycinnamoy1guanidine 2.25 /1 5-tert-butylarino-amiloride 2.01 /2 3-t-butylcinnamoylguanidine 2.00/1 5-bromo-2-fluorocinnamoylguanidine 2.00 / 1 (4-Chlorocinnamoyl)guanidine 1.94/2 -t-butylcinnamoylguanidine 1.86/ 1 2-cyclohexylcinnamoylguanidine 1.83/1 5-Iodoamiloride 1.75/2 -(trans-hept-1-en- 1-yl)cinnamnoylguanidine 1.71 /1 4-Blromocinuamoyl)guanidine 1.6912 (4-Hydroxycinnanoy1)guanidine 1.63/2 -(3-phenylpropanoy)-N'-phenylguanidine 1.57/2 3-Nitrocinnamoyl)guanidine 1.51/2 3-fluorocinnamoylguanidine 1.50/1 2-(1-napthyl)aoetoylguanidine 1.50/1 2-ethyleinnamoylguanidine 1.50/1 5-(N,N-Dimethyl)amiloride hydrochloride - 1.38/2 2-napthoylguanidine 1.38/2 5-(4-fluorophenyl)amiloride 1.38/1 2-(trifluoromethyl)cinnamoylguanidine 1.38 /1 N-(6-Hydroxy-2-napthoyl)-N'-phenyguanidine 1.35/3 (trans-2-Phenyloyclopropanecarbonyl)guanidine 1.34/3 N,N'-bis(3phenylpropanoyl)-N"-phenylguanidine .1.33/3 1-napthoylguanidine 1.32/3 WO 2004/112687 PCT/AU2004/000866 -78 Benzamil hydrochloride 1.32/2 3-methoxy -HMA 1.25/1 4-methylcinnamoylguanidine 1.25/1 4-fluorocinnamoylguanidine 1.25 /1 3,4-(methylenedioxy)cinnamoylguanidine 1.25/1 5-(N,N-hexamethylene)amiloride 1.2/3 N-(cinnamoyl)-N'phenylguanidine 1.19/2 5-(N-Ethyl-N-isopropyl)amiloride 1.07/2 3-methylinnamoylguanidin. 1.00/1 2-methyleinnamoylguanidine 1.00/ 1 2,3,5,6,-tetramethyleinnamoylguanidine 1.00 / 1 trans-3-Furanacryoylguanidine 0.88/2 (4-Methoxycinnamoy1)guanidine 0.88/2 (2-Furanacryloyl)guanidine 0.82/2 (3-phenylpropanoyl)guanidine 0.73/5 2-(2-napthy1)acetoylguanidine 0.71/1 *inamoylguanidine 0.69/3 (2-Methoxycinnamoy)guanidine 0.69/2 [3-(3-Pyridyl)acryloy]guanidine 0.67 /3 4-phenylbenzoylguanidine 0.63/2 ,4-dichlorocinnamolyguanidine 0.63/2 3-Methoxycinamoy)gan-idine .63 /2 -fluorooinnamoylguanidine 0.63/1 4-Phenoxybenzoyl)guanidine 0.57/2 a-Methylcinnamoyl)guanidine 0.50/1 5-(3'-bromophenyl)penta-2,4-dienoylguanidine 0.5/1 (5-Phenyl-penta-2,4-dienoyl)guanidine 0.44/2 (Quinoline-2-carbonyl)guanidine 0.41/1 (Phenylacetyl)guanidine 0.32/3 N,N'-Bis(amidino)napthalene-2,6-dicarboxamide 0.25/2 6-bromo-2-napthoylguanidine 0.2 /1 1-bromo-2-napthoylguanidine 0.25/1 2-chloro-6-fluorocinnamoylguanidine 0,25/1 [(4-Chlorophenoxy-acety]guanidine 0.19/2 Phenamil-methanesulfonate salt 0.13 /2 N-Benzoyl-N'-einnamoylguanidine 0.13/2 N-(2-napthoyl-N'-phonylguanidine 0.07/2 Example 24. SARS Antiviral Assav for testing compounds against replication of SARS coronavirus (SARS-CoV). 5 Compounds were tested against SARS-CoV (Hong Kong strain) using virus plaque purified three times in Vero cells. Stock virus was generated by infecting Vero cells at MOI =x TCIDs per 100 cells. Example 24.1 Screening for anti-viral activity using the virus icrotitre assay WO 2004/112687 PCT/AU2004/000866 -79 Monolayers of Verd cells grown in 25cm 2 flasks were infected at a multiplicity of 1:50 and treated immediately post infection with compounds at two concentrations, 1OuM and 20M. A control infected monolayer remained untreated. Samples of culture media were taken at 48 hours post infection. Two aliquots from 5 each of the samples (titrations 1 and 2) were serially log diluted and 12 replicates of log dilutions -4 to -7 added to cells in microtitre plates. Four days later, wells in the microtitre plates were scored for cytopathic effect (CPE) and the titration values calculated based on the number of CPE positive wells at the 4 dilutions. Control titres were 4.8 and 5.9 TCIDso x 106 (average 5.35 x 106) 10 Example 25: Effect of compounds in SARS CoV antiviral assav Three selected compounds were tested for activity against SARS-CoV according to the method described in example 21. For trans-3-(1 15 napthyl)auryloylguanidine and vinnamoylguanidin0 a decrease in virus titre of approximately 80% was observed at a concentration of OuM and a reduction of approximately 50% was seen to persist at 2pM trans-3-(1-napthyl)acryloylguaidine. Table 8 provides Virus titration data presented as % of a control (SARS CoV grown 20 for 48 hours in the absence of compounds). Table 8 Compound Average Titre Concentration TCID 0 (x (% Name (UM) 10 ) control) cinnamoylguanidine 10 1.3 24 2 4.4 82 trans-3-(1- 10 1.15 22 napthyl)acryloylguanidine 2 2.45 46 6-methoxy-2-naphthoylguanidine 10 5,95 i1 2 6.35 118 Control 0 5.35 100 25 WO 2004/112687 PCT/AU2004/000866 -80 Example 26. Human 229E Coronavirus Synthesis and Purification of a Peptide Corresponding to the 229E-E Protein A peptide corresponding to the full-length 229E- protein (sequence: 5 MFLKLVDDHALVVNVLLWCVVLIVILLVCITI KLCFTCHMFCNRTVYGPI KNVYHIYQSYMHIDPFPKRVIDF; accession number NP_073554) was synthesized manually using FMOC chemistry and solid phase peptide synthesis. The synthesis was done at the Biomolecular Resource Facility (John Curtin School of Medical Research, ANU, Australia) using a Symphony" Peptide Synthesiser from Protein 10 Technologies Inc.(Woburn, MS, USA) according to the manufacturers instructions to give C-terminal amides, the coupling was done with HBTU and hydroxybenzotriazole in N-methylpyrrolidone. Bach of the synthesis cycles used double coupling and a 4-fold excess ofthe amino acids. Temporary a-N Fmoc protecting groups were removed using 20% piperidine in DMF. 15 The crude synthetic peptide was purified using the ProteoPlus tm kit (Qbiogene inc. CA), flowing manufactures instructions. Briefly, the peptides were diluted in loading buffer (60mM Tris-HCI pH 8.3, 6M urea, 5% SDS, 10% glycerol, 0.2% Bromophenol blue, ±100 mM 1-mercaptoethanol) and run on 4-20% gradient polyacrylamide gels (Gradipore, NSW, Australia) in tris-glycine electrophoresis 20 buffer (25 mM Tris, 250 mM glycine, 0.1% SDS). The peptides were stained with gel code blue (Promega, NSW) and the bands corresponding to the fufl-length peptide were excised out of the gel. The gel slice was transferred to the ProteoPLUSTM tube and filled with tris-glycine 25 electrophoresis buffer. The tubes were emerged in tris-glycine electrophoresis buffer and subjected to 100 volts for approximately 1 hour. The polarity of the electric current was reversed for 1 minute to increase the amount of protein recovered. The peptides were harvested and centrifuged at 13, 000 rpm for 1 minute. The purified peptides were dried in a Speedvac and the weight of the final product was used to 30 calculate the yield.

WO 2004/112687 PCT/AU2004/000866 -81 Example 27. 229E-E protein forms ]on channels In planar Hluld bilavers. Lipid bilayer studies were performed as described elsewhere (Sunstrom, 1996; Miller, 1986). A lipid mixture of palmitoyl-oleoyl-phosphatidylethanolamine, palnitoyl-oleoyl-phosphatidylserine and palmitoyl-oleoyl-phosphatidylcholine 5 (5:3:2) (Avanti Polar Lipids, Alabaster, Alabama) was used. The lipid mixture was painted onto an aperture of 150-200 gm in the wall of a I ml deirin cup. The aperture separates two chambers, cis and trans, both containing salt solutions at different concentrations. The cis chamber was connected to ground and the trans chamber to the input of an Axopatch 200 amplifier. Normally the cis chamber contained either 10 500 mM NaCI or 500mMKCl and the trans 50 uM NaCI or 50mM KCL. The bilayer formation was monitored electrically by the amplitude of the current pulse generated by a current ramp. The potentials were measured in the trans chamber with respect to the cis. The synthetic peptide was added to the cis chamber and stirred until channel activity was seen. The currents were filtered at 1000 Hz, digitized at 5000 Hz and 15 stored on magnetic disk. The 229E E synthetic peptide was dissolved in 2,2,2-trifluorethanol (TPE) at 0.05mg/mi to 1 mg/ml. 10 pl of this was added to the cis chamber (1l aqueous volume) of the bilayer apparatus, which was stirred via a magnetic "flea". Ionic currents, indicating channel activity in the bilayer, were typically detected within 15 20 30 min. After channels were detected the holding potential across the bilayer was varied between -100mV and +lOOmV to characterise the size and polarity of current flow and enable the reversal potential to be detennined. In 15 experiments where the cis chamber contained 500mM NaCI solution and the trans chamber contained 50 mM NaCl solution, the average reversal potential 25 of the channel activity was calculated to be 22 ±7 (SEM) mV. In 13 experiments where the cis chamber contained 500mM KCl solution and the trans chamber contained 50 mM KC solution, the average reversal potential of the channel activity was calculated to be 38±4 (SEM) mV. These results indicate that the 229EB protein forms cation selective ion channels that are slightly more selective for K 4 than for 30 Na* ions.

WO 2004/112687 PCT/AU2004/000866 -82 Figure 9 shows examples of raw current data for the 229E B ion channel at various holding potentials (cis relative to trans) in asymmetrical M(0 solutions (500/50 mM). The graph is a representative plot of average bilayer current (pA; y-axis) versus holding potential (mV; x-axis). 5 Example 28. Chemical compounds inhibit the ion channel activity of the 229E E Protein synthetleneptide. To test compounds for their ability to block or otherwise inhibit the ion channel formed by 229E E protein, small aliquots of solutions containing the 10 compounds were added to the aqueous solutions bathing planar lipids in which the peptide channel activity had been reconstituted and the effect of the compound addition on the ionic currents was recorded and measured. Compound stock solutions were typically prepared at 500 mM in DMSO. This solution was further diluted to 50 mM, or lower concentration in 50% DMSO/50% 15 methanol and2 pl of the appropriately diluted compound was added to the cis and/or trans chambers to yield the desired fmal concentration. In the example shown in Figure 10, addition of 100pM cinnamoylguanidine to the cis chamber greatly reduced current flow through the 229E E ion channel. 20 Example. 29. Bacterial Bio-Assay for Screening Potential 229E-CoV E protein Ion Channel-Blocking Drugs. 229E-CoV E-protein Ion Channel inhibits Bacterial Cell growth. 25 A bio-assay of 229E-CoV E-protein function in bacterial cells was developed. A synthetic cDNA fragment encoding 229E-CoV E-protein was cloned into the expression plasmid pPL451, creating a vector in which B protein expression is temperature inducible, as described in Example 4. Inhibition of the growth of E.coli cells expressing E protein at 37*C was observed as an indicator of p7 ion channel 30 function dissipating the normal Na+ gradient maintained by the bacterial cells. 35 WO 2004/112687 PCT/AU2004/000866 -83 Example 30 Compound Screening using the Bacterial Bio-Assay for 229E-CoV E-protein. The halos of g-owth around the site of application of particular drugs - as described in example 14 -were scored as decribed in example 15. 5 Table 9 list the scores fbr ihibition of 229E-CoV E-protein in the bacterial bio-assay. Table 9 229E E protein Inhibition Compound (score) 4-isopropylinnamoylguanidine 4.9 3,4-dichlorocinnamoylguanidine 4.4 3-(trifluoromethoxy)cinnamoylguanidine 4.1 4-t-butylcinnamoylguanidine 4.0 3-isopropylcinnamoylguanidine hydrochloride 4.0 3-t-butylcinnamnoylguanidine 3.9 2--butyloinnamoylguanidine 3.9 trans-3-(1-napthyl)acryloylguanidine 3.7 5 -bromo-2-methoxycinnamoylguanidine 3.6 2,3-difluorocinnamoylguanidine 3.3 3-(2-napthyl)acryoyguanidine 3.0 2-phenylcinnamoylguanidine 3.0 3-phenyloinnamoylguanidine 2.9 3-(cyclohex-1-en-1-yl)cinnamoylguanidine 2.4 4-phenylbenzoylguanidine 2,3 3-(trifluoromethyl)cinnamoylguanicdine 2.3 (4-Phenoxybenzoyl)guanidine 2.3 (trifluoromethyl)cinnamoylguanidine 2.3 2-(cyclohex-1-en-lyl)cinnamoylguanidine 2.3 (4-Bromocinnamoyl)guanidine 2.0 5-(NN-hexamethylene)amiloride 1.9 1-napthoylguanidine 1.9 5-(4-fluorophenyl)amiloride 1.8 (5-Phenyl-penta-2,4-dienayl)guanidine 1,8 (3-Bromocinnamoyl)guanidine 1.7 ,5-dimethyloinnamoylguanidine 1.6 2-(trifluoromethyl)cinnamoylguanidine 1.5 6-methoxy-2-naphthoylguanidine 1.4 (4-Chlorocinnamoyl)guanidine 1.4 (3-Methoxycinnamoyl)guanidine 1.4 5-bromo-2-fluorocinnamoylguanidine 1.4 5-(NN-Dimethyl)amiloride hydrochloride 1.3 WO 2004/112687 PCT/AU2004/000866 i84 cinnamoylguanidine 1.3 (2-Methoxycinnamoyl)guanidine 1.1 (4-Metylcitnaznoyl)guanidine 1.0 4-phenylcinnamoylguanidine 1.0 2,6-dichlorocinnamnoylguanidine 1.0 (2-Bromocimnamoy)guanidine 0.9 2,4,6-trimethyloinnamoylguanidine 0.9 (trans-2-Phenylcyclopropanecarbonyl)guanidine 0.8 (3-Chlorocinnamoyl)guanidine 0.8 2-(1-napthyl)acetoylguanidine 0.8 2-ethyicinnamoyguanidine 0.8 2-cyclohexylcinnamoylguanidine 0.8 (4-Hydroxycinnamoyl)guanidine 0.6 2-ethoxycinnamoylguanidine 0.6 3-methyloinnamoyguanidine 0.5 -methylkinnamoylguanidine 0.5 3-fluorocinnamoylguanidine 0.5 cinnamoylguanidine hydrochloride 0.5 2,3-dimethylcinnamoylguanidine 0.5 2-fluorocinnamoylguanidine 0.4 4-fluorocinmamoylguanidine 0.4 3,4-difluorocinnamoylguanidine 0.4 5-tert-butylamino-amiloride 0.3 2-napthoylguanidine 0.3 N,N-Bis(amidino)napthalene-2,6-dicarboxamiide 0.3 N,N'-Bis(3-phenylpropanoyl)guanidine 0.3 4-methyleinnamoylguanidine 0.3 5-(3'-bromophenyl)penta-2,4-dienoylguanidine 0.3 2,3,5,6,-tetramethylciinamoylguanidine 0.3 3-ethoxycinnamoylguanidine 0.3 N,N-bis(3phenylpropanoyl)-N"-phenylguanidino 0.1 (4-Methoxycinnamoyl)guanidine .0.1 (2-Chlorocinnamoyl)guanidine 0.1 (3-Nitrocinnamoyl)guanidine 0.1 4-ethoxycinnamoylguanidine 0.1 3,4,5-trimethoxycinnamoylguanidine 0.1. 2-(2-napthyl)acetoylguanidine 0.1 IN-(3--heypropanoy)-N'-phenylguanidine 0.1 Example 31: Antiviral Assay for testing compounds against replication of human coronavirus 229E (229E). To determine the antiviral activity of compounds against human coronavirus 229 replication (ATCC VR-740), an assay measuring reduction in the number of WO 2004/112687 PCT/AU2004/000866 -85 plaques formed in monolayers of 229E infected MRC-5 cells (human lung fibroblasts ;ATCC CCL-171) was developed: First, a virus working stock was prepared by amplificationin MRC-5 cells, This was then used to infect confluent monolayers of MRC-5 cells grown in 6-well tissue culture plates by exposure to the virus at an MOI 5 of approx. 0.01 pfh/cell for 1 hour at 35*C in 5%CO2. The infective inoculum was removed and replaced with fresh medium (DMEM supplemented with 10% fetal calf serum) containing various test concentrations of compounds or the appropriate level of solvent used for the compounds (control). Plates were subsequently incubated at 35"C (in 5% C2) for 3 -5 days post infection, after which time culture supernatant 10 was removed and the cells were stained with 0.1% crystal violet solution in 20% ethanol for 10 minutes. Plaques were counted in all wells and the percentage reduction in plaque number compared to solvent control was calculated. Measurements were performed in duplicate to quadruplicate wells. 15 Table 10 Plaque Reduction (%control / # experiments) Compound SuM 2.uM luM -t-butylcinnamoylguanidine 100 /1 100/3 050/3 4-isopropylkinnamoylguanidine 100/1 100/2 057/2 3,4-dichlorocinnamoylguanidine 100/3 099/4 086/3 3 'trifluoromethoxy)cinnamoylguanidine 100 /2 098/4 077/3 2,6-dichlorocinnamoylguanidine 100/1 097 /3 066 /2 7-(cyclohex-1-en lyl)cinnamoylguanidine 100/1 097/3 021/1 2-cyclohexyloinnamoylguanidine 070/1 097/2 089/2' 5-bromo-2 methoxycinnamoylguanidine 100 /2 096/4 088/3 2-phenylcinnamoyguanidine 100 /1 096/ 3 100/1 5-(2'-bromophenyl)penta-2,4 dienoylguanidine 100/2 095/3 079/2 4-butyluinnmnoylguanidine 100/ 1 095/3 084/3 3-phenyloinnamoylguanidine 094/3 077/2 (3-Bromocinnamoyl)guanidine 100 /2 093/3 072 /2 (4-Bromocinnamoyl)guanidine 094 / 1 091 /3 073/2 5-(NN-hexamethylene)amiloride 089 / 091 /2 033/1 trans-3-(1-napthyl)acryloylguanidine 10011 091 /2 064/2 WO 2004/112687 PCT/AU2004/000866 -86 3-(2-napthyl)acryloylguanidine 100/ 1 091 /2 062/2 2,4-dichlorocinnamolyguanidine 100/2 090 / 4 064/3 (2-Nitrocinnamoyl)guanidine 085 /2 090/2 046 /2 3 (trifluoromethyl)cinnamoylguanidine 097/2 089/4 064/3 5-bromo-2-fluorocinnamoylguanidine 100 /1 088/3 063 /2 4-methyloinnamoylguanidine 091 /2 087 /4 063 /2 (3-Chlorocinnamoyl)guanidine 100 /1 086/3 009 /1 (4-Methoxycinnamoyl)guanidine 100 / 1 085 /4 057 /3 (4-Chlorocimnamoyl)guanidine 100 /2 084 /2 051 /2 3-fluorocinnamoylguanidine -095/1 083 /3 051 /2 3-(oyclohex-1-en-1- y)cinnanoylguanidine 100 /1 082/3 063 /2 (a-Methylcinnamoyl)guanidine 023 /1 082 /1 036 /2 2,3,5,6, tetramethylcinnmoylguanidine 098/2 079 /4 064 /3 -fluorocinnamoylguanidine 090 /1 079/3 045 /2 4 (tifluoromethyl)cinnamoylguanidine 100/1 079 /1 052 /1 (3-Nitrocinnamoyl)guanidine 100 / 1 079/1 045 /1 2,5-dimethylkinnamoylguanidine 092/2 078/1 078/1 3-t-butylcinnamoylguanidine 100 /1 077/4 - 030 /3 (3-Methoxycinnamoyl)guanidine 089 /1 075 /2 030/1 3-methylcinnamoylguanidine 095/1 074/3 044 /1 3-isopropyloinnamoylguanidine ydrochloride 089/1 074/3 014/1 (2-Bromooinnamoyl)guanidine 095 /2 072/2 043/2 3-ethoxycinnamoylguanidine 100 / 1 072/3 057 /1 (5-Phenyl-penta-2,4-dienoyl)guanidine 100/1 072 /2 069/1 (2-Chlorocinnamoyl)guanidine 095 /2 072/2 040 /2 4-ethoxycinnamoylguanidine 073 /1 069 /2 057 /1 4-fluorocinnamoylguanidine 100/1 067/3 034 /2 3,4-difluorocinnamoylguanidine 085 /1 065/3 042 /2 N-(3-phenylpropanoyl)-N' phenylguanidine 051/1 064/1 000/1 2,4,6-trimethyloinnamoylguanidine 075 /2 063/3 062 /2 2-methylcinnamoylguanidine 074/2 063/3 053/3 (trans-2 Phenylcyclopropanecarbonyl) guanidine 063/2 022/1 {(E)-3-(4-Dhnethylaminophenyl)-2 methylacryloyl]guanidine 059/1 N-Benzoyl-N'-cinnanoylguanidine 056/1 4-phenylbenzoylguanidine 076 / 1 055 /2 071 /1 trans-3-Furanaeryoylguanidine 055 /2 018/1 (4-Phenoxybenzoyl)guanidine 069/1 054/3 040 /2 (2-Methoxycinnamoyl)guanidine 051 /1 053/2 024 /1 N-amidino-3-amino-5-phenyl-6- 074/2 052/2 038 /1 WO 2004/112687 PCT/AU2004/000866 -87 chloro-2 pyrazinecarboxamide N-(cinnamoyl)-Nphenylguanidine 084 /1 .048 /2 035 / 1 cinnamoylguanidine 095 /2 047 /2 059 / 1 3,4 (methyleanedioxy)cinnamoylguanidine 084/1 046/1 019 / 1 ,N'-Bis(amiidino)napthalene-2,6 dicarboxamide 045/1 .3-dimethylcinnamoylguanidine 073/1 044/2 024/1 5.(3'-bromophenyl)penta-2,4 dienoylguanidine 044/ 1 N,N-Bis(3 phenylpropanoyl)guanidine 041 /1 -methoxy-amiloride 029 /2 039/3 022 /2 2,3-difluorocinnamoylguanidine 036 /1 1-napthoylguanidine 036/1 (3-phenylpropanoyl)guanidine 036 /1 .6-methoxy-2-naphthoylguanidine 49/3 030/4 5-(NN-Dimethyl)amiloride hydrochloride 027/1 2-ethoxycinnamoylguanidine 027/1 2-napthoylguanidine 027/1 3,4,5-trimethoxycinnamoylguanidine 027/1 3-methoxy -HM4A 027/ 1 enzyoylguanidine 026/1 trifluoromethyl)cinnamoylguanidine 022/1 N-amidino-3,5-diamino-6-phynyl-2 pyrazinecarboxanide 022/1 cinnamoylguanidine hydrochloride 020/ 1 Quinoline-2-carbonyl)guanidine 015/3 019/3 006/2 (4-Hydroxycinnamoyl)guanidine 019 /1 -(4-fluorophonyl)amilorid6 018/1 -(1-napthyl)acetoylguanidine 018/1 (2-Furanacryloyl)guanidine 018/1 [3-(3-Pyridyl)acryloyl]guanidine 018/1 N-Cinnamoyl-N',N' dimethylguanidine 015/1 N-(2-napthoyl)-N'-phenylguanidine 011/1 2-(2-napthyl)acetoylguanidine 009/1 N,-bis(3phenylpropanoyl)-N" phenylguanidine 009/1 (Phenylacety1)guanidine . 009 /1 WO 2004/112687 PCT/AU2004/000866 -88 Example 32 Human OC43 Coronavirus 0C43 Antiviral Assay for testing cOmpounds against replication of human coronavirus OC43. To determine the antiviral activity of compounds against human coronavirus 5 0C43 replication (ATCC VR-759), an ELISA assay was developed measuring the release of the viral N-protein into culture supernatants from monolayers of OC43 infected MRC-5 cells (human lung fibroblasts ;ATCC CCL-171): First a virus working stock was prepared by amplification in MRC-5 cells. This was then used to infect confluent monolayers of MRC-5 cells grown in 6-well tissue culture plates by 10 exposure to the virus at an MOI of approx. 0.01 pfu/cell for 1 hour at 35"C in 5%CO2. The infective inoculum was removed and replaced with fresh medium (DMEM supplemented with 10% fetal calf serum) containing various test concentrations of compounds or the appropriate level of solvent used for the compounds (control). Plates were subsequently incubated at 35 0 C (in 5% CO 2 ) for 5 days post infection, 15 after which time culture supernatant was harvested and cellular debris removed by centrifugation at 5000 x g for 10 minutes, For N-antigen detection, 100pl samples of clarified culture supernatant were added to duplicate wells of a 96-well Maxi-Sorb plate; 100yl of RIPA buffer was added per well with mixing and the plate was covered and incubated at 40C overnight to enable protein binding to the plastic wells. 20 The next day, the coating solution was discarded, wells were washed thoroughly with PBST, and blocking of unoccupied protein binding sites was performed by incubation in 1% BSA in PBS for 1.5 hours. The antibody recognising OC43 N-protein was used at 1/800 dilution in PBS (lhr at 374C) and the secondary antibody (goat-anti mouse alkaline phosphatase) was Used for the colour development reaction. Optical 25 density of the wells was read at 405 nm and the effect of compounds detennined by comparison of the level of signal in presence of compound to level of signal from the solvent control. Example 33: Effect of compounds in OC43 antiviral assay 30 Compounds were screened for activity against OC43 replication according to the method described in example 22. Results are shown in Table 11.

WO 2004/112687 PCT/AU2004/000866 -89 Table 11 Virus inhibition at Compound 2.5uM 3-methyleinnamoylguanidine 100 trans-3-(1-napthyl)acryloylguanidine 100 (3-Bromocinnamoyl)guanidine 100 (2-Chlorocinnamoyl)guanidine 96 3,4-dichlorocinnamoyguanidine 90 3-(trifluoromethyl)cinnamoylguanidine 84 (trans-2-Phenylcyclopropanecarbonyl)guanidine 71 4-isopropylcinnamoylguanidine 68 cinnamoylguanidine 57 6-methoxy-2-naphthoylguanidine 47 2,4-dichlorocinnamolyguanidine 36 (4-Chlorocinnamoyl)guanidine 36 5-(N,N-hexamethylene)atniloride, 30 (4-Bromocinnamoyl)guanidine 29 2,6-dichlorocinnamoylguanidine 27 5-bromo-2-methoxycinnamoylguanidine 24 (5-Phenyl-penta-2,4-dienoyl)guanidine - 9 3-(trifiioromethoxy)cinnamoylguanidine 4 2bl innamoylguidine 4 5 Example 34. Mouse Hepatitis Virus (MIV). Synthesis and Purification of a Peptide Corresponding to the MH9V-A59 E Protein. A peptide corresponding to the full-length MHV-A59 E protein (sequence: 10 MFNLFLTDTVWYVGQFIFAVCLMVTIIVVAFLASIKLCIQLCGLCNTL VLSPSIYLYDRSKQLYKYYNEEMRLPLLEVDDI; accession number NP_068673) was synthesized manually using FMOC chemistry and solid phase peptide synthesis The synthesis was done at the Biomolecular Resource Facility (John Curtin School of Medical Research, ANU, Australia) using a Symphony Peptide Synthesiser from 15 Protein Technologies Inc.(Woburn, MS, USA) according to the manufacturers instructions to give C-terminal amides, the coupling was done with HBTU and hydroxybenzofriazole in N-methylpyrrolidone. Each of the synthesis cycles used double coupling and a 4-fold excess of the amino acids. Temporary a-N Fmoc protecting groups were removed using 20% piperidine in DMF.

WO 2004/112687 PCT/AU2004/000866 -90 The crude synthetic peptide was purified using the FroteoPlusTM kit (Qbiogene inc. CA), following manufactures instructions. Briefly, the peptides were diluted in loading buffer (60mM Tris-HC pH 8.3, 6M urea, 5% SDS, 10% glycerol, 0.2% Bromophenol blue, ± 100 mM S-mercaptoethanol) and run on 4-20% gradient 5 polyacrylamide gels (Gradipore, NSW, Australia) in tris-glycine electrophoresis buffer (25 mM Tris, 250 mM glycine, 0.1% SDS). The peptides were stained with gel code blue (Promega, NSW) and the bands corresponding to the Dill-length peptide were excised out of the gel. The gel slice was transferred to the ProteoPLUSTM tube and filled with tris 10 glycine electrophoresis buffer. The tubes were emerged in tris-glycine electrophoresis buffer and subjected to 100 volts for approximately 1 hour. The polarity of the electric current was reversed for 1 minute to increase the amount ofprotein recovered. The peptides were harvested and centrifuged at 13, 000 rpm for 1 minute. The purified peptides were dried in a Speedvac and the weight of the final product 15 was used to calculate the yield. Example 35: MHV-E protein forms ion channels in planar lipid bilayers. Lipid bilayer studies were performed as described elsewhere (Sunstrom, 1996; Miller, 1986). A lipid mixture of palmitoyl-oleoyl-phosphatidylethanolamine, 20 palnitoyl-oleoyl-phosphatidylserine and palmitoyl-oleoyl-phosphatidylcholine (5:3:2) (Avanti Polar Lipids, Alabaster, Alabama) was used. The lipid mixture was painted onto an aperture of 150-200 pm in the wall of a 1 ml delrin cup. The aperture separates two chambers, cis and trans, both containing salt solutions at different concentrations. The cis chamber was connected to ground and the trans chamber to 25 the input of an Axopatch 200 amplifier. Normally the cis chamber contained either 500 mM NaCI or 500mM KC and the trans 50 mM NaCl or 50mM KC1. The bilayer formation was monitored electrically by the amplitude of the current pulse generated by a current ramp. The potentials were measured in the trans chamber with respect to the cis. The synthetic peptide was added to the cis chamber and stirred until channel 30 activity was seen. The currents were filtered at 1000 Hz, digitized at 5000 Hz and stored on magnetic disk.

WO 2004/112687 PCT/AU2004/000866 -91 The MHV E synthetic peptide was dissolved in 2,2,2-trifluorethanol (TPE) at 0.05mg/ml to 1 mg/ml. 10 pl of this was added to the cis chamber (iml aqueous volume) of the bilayer apparatus, which was stirred via a magnetic "flea" Ionic currents, indicating channel activity in the bilayer, were typically detected within 15 5 30 min. After channels were detected the holding potential across the bilayer was varied between -100mV and +100mV to characterise the size and polarity of current flow and enable the reversal potential to be determined. In 14 experiments where the cis chamber contained 500mM NaC1 solution and the trans chamber contained 50 mM NaCl solution, the average reversal potential 10 of the channel activity was calculated to be 49 L1 (SEM) mV. In 11 experiments where the cis chamber contained 500mM KCI solution and the trans chamber contained 50 mM KC1 solution, the average reversal potential of the channel activity was calculated to be 13 ±6 (SEM) mV. These results indicate that the MHIV E protein forms cation selective ion channels that are more selective for Na* than for K ions. 15 Figure 11 shows examples of raw current data for the MHV E ion channel at various holding potentials (cis relative to trans) in asymmetrical NaCL solutions (500/50 mM). The graph is a representative plot of average bilayer current (pA; y axis) versus holding potential (mV; x-axis). 20 Example 36. Chemical compounds Inhibit the ion channel activity of the MHV E protein synthetic Dentide. To test compounds for their ability to block or otherwise inbibit the ion channel formed by MHV E protein, small aliquots of solutions containing the compounds were added to the aqueous solutions bathing planar-lipids in which the 25 peptide channel activity had been reconstituted and the effect of the compound addition on the ionic currents was recorded and measured. Compound stock solutions were typically prepared at 500 mM in DMSO. This solution was further diluted to 50 mM, or lower concentration in 50% DMSO/50% methanol and 2 pl of the appropriately diluted. compound was added to the cis and/or 30 trans chambers to yield the desired final concentration.

WO 2004/112687 PCT/AU2004/000866 -92 In the example shown in Figure 12 below, addition of 100gM cinnamoylguanidine to the cis chamber greatly reduced current flow through the MUV E ion channel. 5 Example 37.Bacterial Bio-Assay for Screening Potential MHV E-protein Ion Channel-Blocking Drugs. 10 MHV E-protein Ion Channel inhibits Bacterial Cell growth. A bio-assay of MHV E-protein function in bacterial cells was developed. A synthetic cDNA fragment encoding MHV E-protein was cloned into the expression plasmid pPL451, creating a vector in which E protein expression is temperature inducible, as described in Example 4. Inhibition of the growth of E.coli cells expressing E protein 15 at 37"C was observed as an indicator of p7 ion channel function dissipating the normal Na+ gradient maintained by the bacterial cells. Example 38. Compound Screening using the Bacterial Bio-Assay for MHV E protein. 20 The halos of growth around the site of application of particular drugs - as described in example 14 -were scored as described in example 15. Table 12 lists the scores for inhibition of MHV E protein in the bacterial bio-assay. 25 Table 12 MHV E protein Inhibition Compound (score) 4-isopropylcinnamoylguanidine 4.5 3-isopropyleinnamoylguanidine hydrochloride 4.2 4-t-butylcinnamoylguanidine 4.1 3-(trifluoromethoxy)cinnamoylguanidine 4.1 3-t-butylinmamoylguanidine 4.0 3,4-dichlorocimamoylguanidine 3.8 2,3-difluorocinnamoylguanidine 3.8 2-t-butylcinnamoylguanidine 3.8 3-phenylcinnamoylguanidine 3.7 2-phenylcinnamoylguanidine 3.4 5-bromo-2-methoxycinnamoylguanidine 3.3 2-(cyclohex-1-en-lyl)cinnamoylguanidine 3.3 WO 2004/112687 PCT/AU2004/000866 -93 3-(trifluoromethyl)cinnamoylguanidine 2.9 -3-(cyclohex-1-en-1-yl)cinnamoylguanidine 2.9 ans-3-(1-napthyl)acryloylguanidine 2.8 4-(trifluoromethyl)cinnamoylguanidine 2.8 3-(2-napthyl)acryloylguanidine 2.8 2-(trifluoromiethyl)cinnamoylguanidine 2.7 (4-Phenoxybenzoyl)guanidine 2.4 (3-Bromocinnamoyl)guanidine 2.4 2,5-dimethyleinnamoy1guanidine 2.3 5-bromo-2-fluorocinnamoylguanidine 2.1 6-methoxy-2-naphthoylguanidine 1.8 4-phenythenzoylguanidine 1.8 (4-Bromocinnamoyl)guanidine 1.8 1-napthoylguanidine 1.7 (5-Phenyl-penta-2,4-dienoyl)guanidine 1.4 (2-Bromocinnamoyl)guanidine 14 (4-Chlorocinnamnoyl)guanidine 1.3 2-methylcinnamoy1guanidine 1.2 2,6-dichlorocinnamoylguanidine 1.2 2,4,6-trimethylcinnamoylguanidine 1.2 5-(N,N-hexamethylene)amiloride 1.1 cinnamoylguanidine 1.1 cinnmnoylguanidine hydrochloride 1,1 (a-Methylcinnamoyl)guanidine 1.0 2,3-dimethylcinnamoylguanidine 1.0 2-cyclohexylcinnamoylguanidine 0.9 -(3-phenylpropanoyl)-N-phenylguanidine 0.8 ,N'-bis(3phenylpropanoyl)-N"-phenylguanidine 0.8 (3-Methoxycinnamoyl)guanidine 0.8 (2-Methoxycimusnoyl)guanidine 0.8 3-fluorocinnamoyguanidine 0,8 2-fluorocinnamoylguanidine 0.8 2,4-dichlorocinnamolyguanidine 0.8 2-ethylcinnamoylguanidine 0.8 (2-Chlorocinnamoyl)guanidine 0.7 (4-Hydroxycinnamoyl)guanidine 0.7 2-ethoxycinnamoylguanidine 0.7 2-napthoylguanidine 0.6 (trans-2-Phenylcyclopropanecarbonyl)guanidine 0.6 5-(N,N-Dimethyl)amiloride hydrochloride 0.5 5-(4-fluorophenyl)amiloride 0.5 3-methylcinnamoylguanidine 0.5 (3-Chlorocinnamoyl)guanidine 0.4 4-methylcinnamoylguanidine 0,4 4-ethoxycionamoylguanidine 0.4 2-(1-napthyl)acetoylguanidine 0.4 WO 2004/112687 PCT/AU2004/000866 -94 3,4-difluorocinnamoylguanidine 0.4 2-(2-napthyl)acetoylguanidine 0.4 2,3,5,6,-tetramethyleinnamoylguanidine 0.4 (4-Methoxycinnamoyl)guanidine 0.3 3,4-(methylenedioxy)cinnamoylguanidine 0.3 3-ethoxycinnamoylguanidine 0.3 4-fluorocinnamoylguanidine 0.2 1-bromo-2-napthoylguanidine 0.2 5-tert-butylamino-amiloride 0.1 (3-Nitrocinnamoyl)guanidine 0.1 3,4,5-trimethoxycinnamoylguanidine 0.1 5-(3'-bromophenyl)penta-2,4-dienoylguanidine . 0.1 5 Example 39. MHV Antiviral Assay for testing compounds against replication of mouse hepatitis virus (MHV). To determine the antiviral activity of compounds against MHV replication (strain MHV-A59: ATCC VR-764), an assay measuring reduction in the number of plaques formed in monolayers of MIV infected L929 cells (ATCC CCL-a) was 10 developed: First, a virus working stock was prepared by amplification in NCTC clone 1469 cells (ATCC CCL-9.1). This was then used to infect confluent monolayers of L929 cells grown in 6-well tissue culture plates by exposure to the virus at an MOI of 0.01 pfu/cell or 1 pfu/cell for 30 minutes at 37"C in 5%CO 2 . The infective inoculumn was removed and replaced with fresh medium (DMEM 15 supplemented with 10% horse serum) containing various test concentrations of compounds or the appropriate level of solvent used for the compounds (control). Plates were subsequently incubated at 37*C (in 5% CO2) for 16 --24 hours post infection, after which time culture supernatant was removed and the cells were stained with 0.1% crystal violet solution in 20% ethanol for 10 minutes. Plaques 20 were counted in all wells and the percentage reduction in plaque number compared to solvent control was calculated. Measurements were performed in duplicate to quadruplicate wells. Example 40. Effect of compounds in MHV antiviral assay. Table 13 provides the results obtained from this study.

WO 2004/112687 PCT/AU2004/000866 Table 13 Percent reduction fn Plaque number I # experiments Compound 20uM 10uM luM 5-(3'-bromopheoyl)penta-2,4-dienoylguanidine N/D 99/2 66/1 5-bromo-2-methoxycinnamoylguanidine NID 100/1 66/ 1 3-phenylcinnamoylguanidine Toxic 86/2 64/3 2,3-difluorocinnamoylguanidine Toxic 92/3 64/2 3-ethoxycinnanoylguanidine 100 /1 89/2 58/ 1 5-(2'-bromophenyl)penta-2,4-dicnoylguanidine Toxic 100 / 1 57/1 cinnamoy1guanidine hydrochloride 85/1 72/2 56 /1 (2-Chlorocinnamoyl)guanidine 95/2 88/3 53/3 cinnamoylguanidine 97/8 88/8 52/7 (4-Bromocinnamoyl)guanidine Toxic) 2 98/3 52/3 (2-Bromocinnamoyl)guanidine 91/2 89/3 52/3 (4-Methoxycinnamoy1)guanidine 98/4 96/4 51 /3 (a-Methylcinnamoyl)guanidine 81 /2 75/3 51 /2 3,4-dichlorocinnamoylguanidine 91/2 96 /1 50/2 2-(cycloh'ex--en-lyl)cinnamoylguauidine N/D 97/1 50/1 3,4-difluorocinnamoylguanidine Toxic 91 /2 50/1 3-t-butyleinnamioylguanidine oxic 94/3 49/2 2-ethoxycinnamoylguanidine 93/2 85/3 48/2 WO 2004/112687 PCT/AU2004/000866 -96 trans-3-Furanacryoylguanidine 70/1 65 / 1 48/1 N-amidino-3-amino-5-hexamethyleneimino-6- 84/1 52/2 48 /1 phenyl-2-pyazineoarboxanide (2-Nitroainnamoyl)guanidine 97/1 77/2 47/1 4-(trifluoromethyl)cinnamoylguanidine 97/3 95 /3 46/3 3,4-(methylenedioxy)cinnamoylgnanidine 93/3 82/3 45/3 5-(N-Methyl-N-isobutyl)amiloride 92/ 1 85/1 44/2 (4-Chlorocinnamoyl)guanidine 97/2 88/2 43/3 2,4-dichlorocinnamolyguanidine 76/1 73 / 1 43 / 1 N-(3-phenylpropanoyl)-N'-phenylguanidine 80/1 65/1 43/1 (3-Nitrocinnwnoyl)guanidine 95 /2 77/3 42/3 2-phenylcinnamoylguanidine N/D 100/1 42/1 4-isopropylcinnamoylguanidine 95 /3 93 /3 41 /3 3-(tdfluoromethoxy)cinnamoylguanidine 100/1 90/3 41/2 3-(trifluoromethyl)cinnamoylguanidine '98/1 83'/1 40/1 (4-Nitrocinnamoyl)guanidine 97/1 75 /3 40 3 3-(2-napthy1)acryloylguanidine 93 /1 93/1 40/1 4-ethoxycinnamoylguanidine 96 /1 92/1 40/ 1 2,6-dichlorocinnamoylguanidine 91 /1 70 /1 40/1 2,5-dimethylcinnamoylguanidine 95 /3 91/3 39/3 (3-Bromocinnamoyl)guanidine 95 /2 90/3 39/3 (3-Chlorocinnamoyl)guanidine 94/1 86/2 39/2 WO 2004/112687 PCT/AU2004/000866 -97 3-methylcinnamoylguanidino 90 /1 88/ 1 39 1 (3-Methoxycinnamoyl)guanidine 92 /2 87/2 3713 2-t-butylcinnamoylguanidine N/D 98/2 37/1 [(E)-3-(4-Dimethylaminophenyl)-2- 56/1 5/1 37/1 methylacryloyl]guanidine 56 / 1 4/ 7/ ,N'-bis(1-napthoyl)guanidine 58/1 52/2 35/1 3-methoxy -HMA 15/1 31/1 35/1 5-tert-butylamino-amiloride 89 /4 84/4 34/4 trans-3-(1-napthy1)acryloylguanidine 95/2 86 / 3 34/3 6-methoxy-2-naphthoylgnanidine 88/3 56/3 34/3 2-napthoylguanidine 67/2 36/2 34/2 2-ethylcinnamoylguanidine 96/ 1 81/2 34/1 2,3-dimethylcinnamoylguanidine 95/ i 79 /2 34/1 N'-Cinnamoyl-NN'-dipheny1guanidine 97/1 72/2 34/1 3-isopropyleinnamoylguanidine hydrochloride NID 99 /2 32/1 (4-Phenoxybenzoyl)guanidine 73/1 6511 32 / 1 (trans-2-Phenylcyclopropanecarbonyl)guanidine 77/2 64/2 31 /2 3-fluorocinnamoylguanidine 100 / 1 93 /2 31 / 1 5-bromo-2-fluorocinnamoylguanidine Toxic 81 /2 31 /1 N,N'-bis-(cinnamoyl)-N"-phenylguanidine 16/1 38/ 2 31(1 3-quinolinoylguanidine 27/1 36/2 30/1 2,4,6-trimethylcinnamoylguanidine 91 /2 61/3 27/2 11 WO 2004/112687 PCT/AU2004/000866 -98 1-brorno-2-napthoylguanidine 31/1 27/2 27/1 N-amidino-3,5-diamino-6-phynyl-2- 53 /1 39/2 25/1 pyrazinecarboxamide N-Cinnamoyl-N',N'-dimethylguanidine 92/2 65/3 24/2 (2-Methoxycinnamoyl)guanidine 90/2 85/2 23/2 2-(2-napthyl)acetoylguanidine 52/1 20/2 23 /1 4-phenylcinnamoylgnanidine 53/ 1 36/1 1 /3 (3-(3-Pyridyl)acryloyl]guanidine 81 /2 73/2 21 /2 3,4,5-trimethoxycinnamoylguanidine 84/ 1 84/1 21/1 4-methylcinnamoylguanidine 93/ 1 89/ 1 20/1 4-fluorocinnamoylguanidine 86/1 83 / 1 20/1 -methyloinnamoylguanidine 1 / 1 82/ 1 20/1 6-bromo-2-naphoylguanidine 65/1 3712 19/1 5-(N,N-Dimethy)amiloride hydrochloride 42.14 714 17/4 (5-Phenyl-penta-2,4-dienoyf)guanidine 27/1 24 /1 17/1 2-cyclohexylcinnamoylguanidine 100/1 74/2 16 /1 5-(4-fluorophenyl)amiloride 4 /1 25/ 1 16 /1 Benzyoyguanidine 22/1 39/2 14/1 N-Benzoyl-N'-einnamoylguanidine 0/1 0/ 1 14/1 5-(N,N-hexamethylene)amiloride 84/2 89/ 1 13 /2 N-(cinnamoyl)-N'phenylguanidine 83/1 88 / I 13 /1 (4-Hydroxycinnamoyl)guaniidine 19 /1 15 /1 13/1 WO 2004/112687 PCT/AU2004/000866 -99 2-(trifluoromethyl)cinnamoylguanidine 19 / 1 1511 13/ 1 (Quinoline-2-cafbonyl)guanidine 86/1 84/1 12 1 2-(1napthyl)acetoylg*anidine -19 / 1 02/1 11 / 1 2-chloro-6-fluorocinnamoylguanidine 100 /1 84/2 9/1 N-amidino-3-mnino-5-phenyl-6-chloro-2- 20/1 20/2 9/1 pyrazinecarboxamide 4-phenylbenzoylguanidine 32/1 24/1 5/1 ,N'-bis(2-napthy)guanidine 511 3 / 2 3/1 (Phenylacetyl)guanidine 35 /1 2/1 3/ 1 1-napthoylganidine 71/3 62/3 2/3 NN'-bis(3phenylpropanoyl)-N"-phenylguanidine 67/3 0/4 1/3 3-hydroxy-5-hexamethylencimino-amiloride 16/1 22 2 1 / 1 2'4 DichloroBenzamil HC1 1212 0/3 0/3 2,3,5,6,-tetramethylcinnamoylguanidine N/D 68 /2 0/1 Benzamilhydrochloride 0/1 26/1 0/1 6-lodoamiloride 28/1 21/1 0/1 ,N'.Bis(amidino)napthalene-2,6-dicarboxamide 19/ 1 16/1 0/-1 - [(4-Chlorophenoxy-acetyl]guanidine 19/1 16/1 0/1 (3-phenylpropanoyl)guanidine 51 / 1 03 /1 0/1 -fluorocinnamoylguanidine 76/ 1 73 /1 6-bromo-2-napthoylguanidine 43/1 (2-Furmnacryloyl)guanidine 67 /2 63 /2 -3/2 WO 2004/112687 PCT/AU2004/000866 -100 N-(6-Hydroxy-2-napthoyl)-N'-phenylguanidine 43/1 39/1 -5/1 Amiloride.HCI 21/1 18/1 -5/1 3-(trans-hept-1-en-1-yl)cinnamoylguanidine T/I 23(T)/ 1 -6 /1 3-methoxy-amiloride 60/2 47/3 -7/2 NN'-Bis(3-phenylpropanoyl)guanidine 41 /3 30/4 -813 3-(cyclohex-1-en-1-yl)cinnamoylguanidine T/1 2/2 19/1 Exraple 41. Porcine Respiratorv Coronavirus (PRCV) Antiviral Assay for testing compounds against replication of porcine respiratory 5 coronavirus (PRCV). To determine the antiviral activity of compounds against porcine respiratory coronavirus replication (ATCC VR-2384), an assay measuring reduction in the number of plaques formed innmonolayers of PRCV infected ST cells (procine fetal testis cell line, ATCC CRL-1746) was developed: Confluent ST cells in 6 well 10 plates were infected with a quaternary passage of porcine respiratory virus (PRCV) strain AR310 at three dilutions 10, 5O-1 and 10-2 in PBS to provide a range of plaques numbers to count. 1 00ul of diluted virus was added per well in a. volume of iml of media. Plates wereincubated for one hour on a rocking platform at room temperature to allow virus to adsorb to cells. The viral supernatant was removed and 15 2m/well of overlay containing 1% Seaplaque agarose in lx MEM, 5% FCS was added to each well. Compounds to be tested were added to the overlay mixture by diluting the compounds from frozen stock to a concentration so that the same volume of compound/solvent would be added to the overlay for each concentration of compound. The volume of compound/solvent never exceeded 0.07% of the volume 20 of the overlay. The solvent used to dissolve compounds was DMSO and methanol mixed in equal proportions. Compounds were tested for anti-plaque forming activity at four concentrations, 0.luM, luM, 1OuM and 20uM. Either duplicates or quadruplicates were performed at each concentration. Controls were performed WO 2004/112687 PCT/AU2004/000866 -101 where the same volume of solvent was added to the overlay. The overlay was allowed to set at room temp for 20 mins. The plates were then incubated at 37*C for 2 days. The monolayers were then fixed and stained overnight at room temperature by adding 1ml/well of 0.5% methylene blue, 4% formaldehyde. Overlay agarose and 5 stain was then rinsed off to visualize stained and fixed monolayer Example 42: Effect of compounds in PRCV antiviral assay Compounds were screened for activity against PRCV replication according to i0 the method described in example 29. Table 14 provides EC50 values for some tested compounds. Table 14 Compound ECS (uM) 5-(NN-hexamethylene)amiloride 0.06 &methoxy-2-naphthoylguanidine 0.04 cinnamoylguanidine 0.08 N-(3-phenylpropanoy)-N'-phenylguanlidine 19 3-methyleinnamoy1guanidine 1.43 (3-Bromocinnamoyl)guanidino 11.2 (trans-2-Phenylcyclopropanecarbonyl)guanidine 17.2 trans-3-(1-napthy1)aoryloylguanidine 19.1 2-(2-napthy1)acetoylguanidine 119.6 15 Example 43. Bovine Coronaviras. Autiviral Assay for testing compounds against replication of bovine coronavirus 20 To determine the antiviral activity of compounds against bovine coronavirus replication (ATCC VR-874), an assay measuring reduction in the number of plaques formed in monolayers of BCV infected MDBK cells (bovine Iddney cell line ;ATCC CCL-22) was developed: Confluent MDBK cells in 6 well plates were infected -with a secondary passage of BCV with serially diluted virus diluted to 10-3 5- and 104 in 25 PBS to provide a range of plaques numbers to count. 100ul of diluted virus was WO 2004/112687 PCT/AU2004/000866 -102 added per well in a volume of 1ml of media. Plates were incubated for one hr to allow virs to adsorb to cells. The viral supernatant was removed and 2ml/well of overlay containing 1% Seaplaque agarose in lx MEM, 5% FCS was added to each well, Compounds to be tested were added to the overlay mixture by diluting the 5 compounds from a 0.5M frozen stock to a concentration so that the same volume of compound/solvent would be added to the overlay for each concentration of compound. The volume of compound/solvent never exceeded 0.07% of the volume of the overlay. The solvent used to dissolve compounds was DMSO.and niethanol mixed in equal proportions. Compounds were tested for anti-plaque forming activity 10 at four concentrations, 0.luM, luM, 1OuM and 2OuM. - Quadruplicates were performed at each concentration. Controls were performed where the same volume of solvent was added to the overlay. The overlay was allowed to set at room temp for 20 mins. The plates were then incubated at 37*C for 7 days. The monolayers wore then fixed and stained by adding 1ml/welI of 0.5% methylene blue, 4% 15 formaldehyde.. Example 44: Effect of compounds in BCV antiviral assay Compounds were screened for activity against BCV replication according to the method described in example 31. Table 15 provides EC50 values for some tested 20 compounds. Table 15 Compound EC50 uM (3-Bromocinnamoyl)guanidine 3 3-(trifluoromethyl)cinnamoylguanidino 3 6-methoxy-2-naphthoylguanidine. 9 5-(NN-hexamethylene)amilorde 9 trans-3-( -napthyl)acryloy1guanidine 13 cinnamoylguanidine 42 (5-Phenyl-penta-2,4-dienoyl)guanidine 95 2-(2-napthyl)acetoylguanidine 99 (trans-2-Phenylcyclopropanecarbonyl)guanidine 109 N-(3-phenylpropanoyl)-N'-phenylguanidine 150 4-phenylbenzoylguanidine 190 WO 2004/112687 PCT/AU2004/000866 -103 Etxample 45 Hepatitis C Virus Ion channel activity of Hepatitis C virus P7 Protein Testing of a Synthetic P7 Peptide for channel activity in artificial lipid bilavers A peptide mimicking the protein P7 encoded by the hepatitis C virus (HCV) 5 was synthesised having the following amino acid sequence: ALENLVILNAASLAGTHGLVSFLVFFCFAWYLKGRWVPGAVYAFYGMWPLL LLLLALPQRAYA Lipid bilayer studies were performed as described elsewhere (Miller, 1986). A lipid mixture of palmitoyl-oleoylphosphatidylethanolamine, paimitoyl-oleoyl 10 phosphatidylserine and palmitoyl-oleoyl-phosphatidylcholine (5:3:2) (Avanti Polar Lipids, Alabaster, Alabama) was used. The lipid mixture was painted onto an aperture of 150-200 um in the wall of a 1 ml dekin cup. The aperture separates two chambers, cis and trans, both containing salt solutions at different concentrations. The cis chamber was connected to ground and the trans chamber to the input of an Axopatch 15 200 amplifier. Normally the cis chamber contained 500 mM KCJ and the trans 50 mM KC1. The bilayer formation was monitored electrically by the amplitude of the current pulse generated by a current ramp. The potentials were measured in the trans chamber with respect to the cis. The protein was added to the cis chamber and stirred until channel activity was seen. The currents were filtered at 1000 Hz, digitized at 20 2000 Hz and stored on magnetic disk. The P7 peptide was dissolved in 2,2,2 trifluorethanol(TEE) at l0mg/ml. 10 ul of this was added to the cis chamber of the bilayer which was stirred. Channel activity was seen within 15-20 min. When the P7 peptide was added to the cis chamber and stirred, channel activity was recorded. The potential in the trans chamber was -80 mV and the currents are 25 downwards, The currents reversed at +50 mV close to the potassium equilibrium potential in these solutions indicating that the channels were cation-selective. The amplitude of the open-channel peak is 1.7 pA corresponding to a channel conductance of about 14 pS. In most experiments, "single channels"had a much larger size, presumably because of aggregation of the P7 peptide. The currents 30 reversed at about +40 mV in this experiment. In some experiments the solution in the WO 2004/112687 PCT/AU2004/000866 -104 cis chamber was 150 mM KC and 15 mM KClin the trans chamber. The P7 peptide again produced currents that reversed. Similar results were obtained when both chambers contained NaC. Currents 5 recorded in an experiment when the cis chamber contained 500 mM NaCl and the trans chamber 50 mM NaCL.. Again the currents reversed between +40 and +60 mV, close to the Na equilibrium potential indicating that channels were much more permeable to Na than to K* 10 The channels formed by the P7 peptide were blocked by 5-(NN hexamethylene) amiloride (HMA). Addition of the P7 peptide produced channel activity. Addition of 2 Al of 50 yM HMA to the cis chamber followed by stining resulted in disappearance of the 15 channel activity. Block of channel activity produced by the P7 peptide with 100 pM HMA was recorded in 4 experiments. In 2 experiments, sodium channels (500/50) were blocked by 500 jAM HMA When 10 mM CaC was added to the cis chamber (K solutions) the reversal 20 potential of the currents produced by P7 peptide shifted to more negative potentials indicating a decrease in the PK/Pcm ratio. When the cis chamber contained 500 mM CaC 2 and the trans chamber 50 mM CaC 2 , both positive and negative currents were seen at potentials around +20 mV and 25 it was not possible to determine a reversal potential. Example 46. Recombinant Expression of HCV p7 protein. Two cDNA fragments, each encoding the same polypeptide corresponding to the amino acid sequence of the HCV-Ja p7 protein, were synthesised commercially 30 by GeneScript. The two cDNAs differed in nucleotide sequence such that in one cDNA ("oDp7.coli") the codons were optimised for expression of the p7 protein in. E.coll while in the other cDNA ("cDp7.mam)" codons were biased for expression in WO 2004/112687 PCT/AU2004/000866 -105 mammalian cell lines. cDp7.coli was cloned into the plasmid pPL451 as a BamHI/EcoRI fragment for expression in E.coli. eDp7.mam was cloned into vectors (for example, pcDNA3.1 vaccinia virus, pfastBac-1) for expression ofp7 in mammalian cell lines. 5 Example 47. Role of p7 in enhancement of Gag VLP Budding. The budding ofvirus-like particles (VLP) from cultured HeLa cells results from the expression of retroviral Gag proteins in the cells and co-expression of snall viral-ion channels, such as M2, Vpu and 6K, with the Gag protein enhances budding. 10 Interestingly, the viral ion channels can enhance budding ofheterologous virus particles. Therefore, to assess budding enhancement by p7 it was co-expressed with the HIV-1 Gag protein in HeLa cells, and VLP release into the culture medium was measured by Gag ELISA. To achieve this, the plasmids pcDNAp7 (pc DNA3.1 pcDp7.mam as described in Example 20, p 7 expressed under control of the T7 15 promoter) and pcDNAGag (HIV-1 Gag protein expressed under control of the T7 promoter) were cotransfected into HeLa cells infected with the vaccinia virus strain VTF7.3 (expresses T7 RNA polymerase) and culture supernatants were collected for ELISA assay after 16 hours incubation. Example 48. Assay of the ability of compounds to inhibit p7 ion channel 20 functional activity. The two methods of detecting p7 ion channel fhnctional activity, described in Examples 33-35, were employed to assay the ability of compounds to inhibit the p7 channel. In the case of Example 33, compounds were tested for their ability to inhibit p7 channel activity in planar lipid bilayers. In the case of Example 35 compounds 25 were tested for their ability to reduce the number ofVLPs released from cells expressing both p7 and HIV-1 Gag. 30 WO 2004/112687 PCT/AU2004/000866 -106 Example 49. Bacterial Bio-Assay for Screening Potential HCV 7 protein Ion Channel Bloeking Drugs. HCV p7 Ion Channel inhibits Bacterial Cell growth. 5 A bio-assay ofp7 fumction in bacterial cells was developed. The p7-encoding synthetic cDNA fragment cDp7.coli was cloned into the expression plasmid pPL451, creating the vector pPLp7, in which p7 expression is temperature inducible, as described in Example 4. Inhibition of the growth of E.coli cells expressing p7 at 37*C was observed as an indicator ofp7 ion channel function dissipating the normal 10 Na+ gradient maintained by the bacterial cells. Example 50 Compound Screening using the Bacterial Bio-Assay for HCV p7 protein. The halos of growth around the site of application of particular dmgs - as described 15 in example 14-were scored as decribed in example 15. Table 16 lists the scores for inhibition of HCV p7 protein in the bacterial bio-assay. Table 16 HCV p7 protein Inhibition (score /# of times Compound tested) 2,3-dimethyleinnamoylguanidine 3.88/2 2,4,6-trimethylcinnamoylguanidine 3.75/1 5-bromo-2-fluorocinnamoylguanidine 3.73/6 (4-Bromocinnamoyl)guanidine 3.47/4 2,5-dimethyloinnamoylguanidine 3.43/4 3-(trifluoromethy)cinnamoylguanidine 3.34/3 4-(trifluoromethyl)cinnamoylguanidine 3,33/5 6-methoxy-2-naphthoylguanidine 3.33/3 (2-Chlorocinnamoy1)guanidine 3.31/6 (4-Chlorocinnamoyl)guanidine 3.16/4 (2-Bromocinnamoyl)guanidine 3.00 /3 2,6-dichlorocinnamoylguanidino 3.00/3 (3-Bromocinnamoyl)guanidine 2.92/3 (3-Chlorocinnamoyl)guanidine 2.75/3 2-(trifluoromethyl)cinnamoylguanidine 2.63/3 (4-Phenoxybenzoyl)guanidine 2.63/1 WO 2004/112687 PCT/AU2004/000866 -107 3,4-dichlorocinnamoylguanidine 2.59/3 4-isopropyloinnamoylguanidine 2.51/2 tra-3-(1-napthyl)acryloylguanidine 2.44/2 4-t-butylcinnamoylguanidine 2.42/2 2-t-butylcinnamoy1guanidine 2.36/2 2-ethyloinnamoylguanidine 2.36/2 4-methyloinnamoylguanidine 2.32/2 5-bromo-2-methoxycinnamoylguanidine 2.32/2 3-(trifluoromethoxy)cinnamoylguanidine 2.26/2 2-cyclohexyloinnamoy1guanidine 2.26/2 1-napthoylguanidine 2.25/1 3-t-butylcimanoylguanidine 2.23/2 4phenylbenzoylguanidine 2.19 /2 5-Phenyl-penta-2,4-dienoyl)guanidine 2.13/1 -(cinnamoy)-N'pheny1guanidine 2.13/1 -isopropylcinuamoylguanidine hydrochloride 2.00/1 enzamil hydrochloride 2.0/1 N-(3-phenylpropanoyl)-N'-phenylguanidine 2.0/1 N,N'-bis(3phenylpropanoyl)-N"-phenylguanidine 2.0/1 3-(2-napthy)acryoyguaWidine 1.93 /2 5-(N-Methyl-N-isobutyl)amiloride 1.88/1 2'4 DichloroBenzamil HCI 1.88/1 5-tert-butylanino-aniloride 1.88/1 5-(N-Ethyl-N-isopropy)amiloride 18811 (4-Methoxycinnamoyl)guanidine 1.88/1 -fluorocinnamoylguanidine 1.86/1 3-Nitrocinnamoyl)guanidine 1.71 /1 -ethoxycinnamoylgnanidine 1.63/1 4-tHydroxycinnamoy)guanidine 1.63/1 trans-2-Phenyloyclopropanecarbonyl)guanidine 1.63/1 -ethoxycinnamoylguanidine 1.63/1 ,3,5,6,-tetramethylinnamoylguanidine 1.51 /2 -phenyloinmamoylguanidine 1.5/1 s-3-Furanaeryoylguanidine 1.38/1 N-(6-Hydroxy-2-napthoy1)-N'-phenylguanidine 1.38/1 (2-Furanacryloy)guanidine. 1.38/1 3-(cyclohex-1-en-1-y1)innamoylguanidine 1.32/2 cinnamoylguanidine hydrochloride 1.32/2 5-(N,N-hexamethylene)amiloride 128 /4 2,3-difluorocinnamoylguanidine 124/1 2-(1-napthyl)acetoylguanidiue 1.14/1 (a-Methyloinnamoyl)guanidine 1.14/1 2-Nitrooinnamoyl)guanidine 1.14/1 -Iodoamiloride 1.13/1 3,4-(methylenedioxy)cinnamoylguanidine 1.13/1 -ethoxycinnamoylguanidine 1.00/1 innamoylguanidine 1.00/1 -phenylcinnamoylguanidine 100/1 WO 2004/112687 PCT/AU2004/000866 -108 2-(cyclohex-1 -en-1y1)cinnamoylguanidine 1.00/1 2-napthoylguanidine 1.0/3 3-phenylcinnamoylguanidine 1.0/1 5-(N,N-Dimethy1)amiloride hydrochloride 1.0/1 5-(4-fluorophenyl)amiloride 1.0/1 3-Methoxycinnamoyl)guanidine 1.0/1 2finorocinnamoylguanidine 1.0/1 5-(3'-bromophenyl)penta-2,4-dienoylguanidine 1.0/1 [(4-Chlorophenoxy-acety1]guanidine 1.0/1 (3-phenylpropanoyl)guanidine 1.0/1 2-chloro-6-fluorocinnamoylguanidirie 0.88/1 3-fluorocinnamoylguanidine 0.8611 2-methy1cinnamoylguamidine 0.75/1 (2-Methoxycinnamoy1)guanidine 10.75/1 1 -bromo-2-napthoylguanidine 0:5/1 3,4,5-trimethoxycinnamoylguanidine 0.71/1 3-methylcinnamoylguanidine 0.63 /1 3-(trans-hept-1-cn-1-y)cinnamoylguanidine 0,50/1 Amiloride.HCl 0.5/2 Phenamil methanesulfonate salt 0.5 /1 2,4-dichlorocimamolyguanidine 0.38/1 (4-Nitrocinnamoyl)guanidine 0,25/1 3,4-difluorocinnamoylguanidine 0.13/1 [(E)-3-(4-Dimethylaminopheny1)-2 methylacryloyl]guanidine 0.03/4 Example 51: Equine Arteritis Virus (EAV) Antiviral Assay for testing compounds against replication of equine arteritis virus (A) 5 To detennine the antiviral activity of compounds against EAV replication (strain Bucyrus; ATCC VR-796), an assay measuring reduction in the number of plaques formed in monolayers of EAV infected BHK-21 cells (ATCC CCL-1O) was developed: A virus stock was amplified in RK-13 cells (ATCC CCL-37) and this was then used to infect confluent monolayers of BHK-21 cells grown in 6-well tissue. 10 culture plates by exposure to the virus at an MO of 5X10 3 pfu/cell for 1 hour at 37*C5% 002. The infective inoculum was removed and nd the cells were overlayed with a 1% sea plgque overlay (Cambrex Bio Science) in MEM containing 10% FCS containing and 10, 5 or 1 pM of compounds to be tested or the appropriate level of solvent used for the compounds (control). Plates were subsequently incubated at 15 37*C (in 5% CO 2 ) for 3 days post infection, after which time culture supernatant was removed and the cells were stained with 0.1% crystal violet solution in 20% ethanol WO 2004/112687 PCT/AU2004/000866 -109 for 10 minutes. Plaques were counted in all wells and the percentage reduction in plaque number compared to solvent control was calculated. Measurements were performed in duplicate to quadruplicate wells. 5 Example 52: Effect of compounds in EAV antiviral assay Compounds were screened for activity against EAV replication according to the method described in example 35. Results expressed as IC50 values are shown in Table 17. 10 Table 17 Compound IC50 5-(N,N-hexamethylene)amiloride 7.5 pM (3-Bromocinnamoyl)guanidine 10,pM trans-3-(1-napthy)acryloylganidine 7.5 pM 2-t-butyleinnamoylguanidine 1 pM 2-(eyclohexl -en-1yl)cinnamoylguanidine 10 pM Example 53 Denue Flavivirus Peptide Synthesis of Dengue virus M Protein 15 The C- terminal 40 amino acids ofthe M protein of the Dengue virus type 1 strain Singapore S275/90 (Fu et al 1992) (ALRHPGFTVIALFLAHAIGTSITQKGUFILLMLVTPSMA) was synthesised using the Fmoc method. The synthesis was done on a Symphony Peptide Synthesiser form Protein Technologies Inc (Tucson, Arizona) as used to give C-terninal amides, the 20 coupling was done with BBTU and hydroxybenzotriazole in N-methylpyrrolidone. Each of the synthesis cycle used double coupling and a 4-fold excess of the amino acids. Temporary c-N Fmoc-protecting groups were removed using 20% piperidine in DMF. Example 54. Incorporation of Dengue M virus protein into lipid bilavers. 25 Lipid bilayer studies were performed as described elsewhere (Sunstrom, 1996; Miller, 1986), A lipid mixture of palrnitoyl-oleoyl-phosphatidylethanolamine, WO 2004/112687 PCT/AU2004/000866 -110 palmitoyl-oleoyl-phosphatidylserine and palmitoyl-oleoyl-phosphatidylcboline. (5:3:2) (Avanti Polar Lipids, Alabaster, Alabama) was used. The lipid mixture was painted onto an aperture of 150-200 pm in the wall of a I ml delrin cup. The aperture separates two chambers, cis and trans, both containing salt solutions at different 5 concentrations, The cis chamber was connected to ground and the trans chamber to the input of an Axopatch 200 amplifier. Normally the cis chamber contained 500 mM KCJ and the trans 50 mM KCI. The bilayer formation was monitored electrically by the amplitude of the current pulse generated by a current ramp. The potentials were measured in the trans chamber with respect to the cis. The protein was added to the 10 cis chamber and stirred until channel activity was seen. The currents were filtered at 1000 Hz, digitized at 5000 Hz and stored on magnetic disk, The dengue virus M protein C-terminal peptide (DMVC) was dissolved in 2,2,2 trifluorethanol (TFE) at 0.05mg/ml to I mg/ml. 10 p1 of this was added to the cis 15 chamber of the bilayer which was stirred. Channel activity was seen within 15-30 min, Example 55: Hexamethylene amiloride (HMA) to Inhibits ion channel activity of the dengue virus M protein C-terminal Deptide. Solutions of 50 mM HMA were prepared by first making a 500 mnM solution 20 in DMSO. This solution was further diluted to 50 mM HMA using 0.1 M HCL. 2 pl of the 50 mM HMA was added to the cis chamber after channel activity was seen. The cis chamber contained 1 ml of solution making the final concentration of HMA 100 pM. 25 Example 56. Antiviral Assay for testing compounds against Effects of Dengue flavivirus against evtoproliferation. Compounds were tested at 10, 5, 2.5, 1.25 and 0.625 pM for activity against Dengue I strain Hawaii using a cytoproliferation assay which measures the effect of dengue virus infection on proliferation of LLC-MK2, rhesus macaque monkey kidney 30 cells. The LLC-MK2 cells were infected with a predetermined amount of virus, titrated such that cell proliferation in infected cultures would be significantly reduced WO 2004/112687 PCT/AU2004/000866 -111 compared to uninfected controls. The infected cells were then plated at 1.5x103 cells per well in a 96 well plate. Negative controls (no virus, no experimental compound), positive controls (virus, no experimental compound), and cytotoxicity controls (experimental compound, no virus) were run with each drug assay. The cultures were 5 allowed to grow for 7 days and then Alamar Blue, a fluorescent dye that measures the metabolism of the cultures (red/ox), was added to each culture and the fluorescence value for each culture was measured. The negative control without experimental compound or virus was fixed at 100%. The positive controls and the cultures with compound were scored by calculating their average fluorescence as a percentage of 10 the negative control. At least six replicate wells were measured for each experimental condition. Example 57 Effect of compounds in Dengue antiviral assay: 15 Table 18 Drug Antiviral Cono. Percent of Drug piM Negative Control cinnaoylguanidine Negative control 0 NA Positive control 0 76.5% 10 17.1% 5 38.6% 2.5. 58.3% 1.25 72.6% 0.625 76.6% (2-chlorocinnamoyl)guanidie Negative control 0 NA Positive control 0 80.3% 10 8.4% 5 7.7% 2.5. 22.7% 1.25 52.5% 0.625 64.3% Trans-3-(1-naphth yl)acryloylguanidine Negative control 0 NA Positive control 0 80.4% 10 6.8% WO 2004/112687 PCT/AU2004/000866 -112 5 12.4% 2.5. 38.7% 1.25 73.7%' 0.625 77,7% N.A. - not applicable Example 58: Positive correlation between Bacterial Assay and Anti-viral Assays 5 Example 58.1 Positive correlation between Vpu Bacterial Assay and anti-RIEV-1 Data. A correlative study was performed to measure correlation between the activity scores assigned to compounds tested in the Vpu bacterial assay and the ability of these compounds to inhibit HIV-1 in the anti-viral assay. 10 Example 58.2. Methodology The p24-antigen data for twelve compounds representing various substituted acyl guanidines was compared with the activity scores obtained for those compounds in the Vpu bacterial assay. The data from each assay was initially rank ordered for effectiveness. The rank order fbr the Vpu bacterial assay was determined from all 15 activity scores, the highest score indicating the greatest effectiveness. The rank order for the anti-HV-1 assay was determined based on the overall average value of p24 antigen measured in culture supernatants at all of the drug concentrations tested, with the lowest score indicating the greatest effectiveness. The two rank orders generated were then compared statistically by generating the Spearman's Rank correlation 20 coefficient. Example 58.3. Results and Conclusion The Spearman's correlation coefficient was 0.785 which, by comparison with a statistical table of critical values (for n12), indicates that the two rank orders are 25 significantly positively correlated (P<0.01) (Table 19a). In addition, a different comparison of the Vpu Bacterial assay rank order with a yes/no score for whether the anti-viral data indicated a p24 reduction of at least one order of magnitude, aligned the 'yes' group of compounds with the top 6 compounds by the bacterial assay (Table 19b).

WO 2004/112687 PCT/AU2004/000866 -113 These results are indicative that a positive correlation exists between baoteaial assays and the antiviral assays as performed-~according to the present Invention. The bacterial assay may therefore be a useful tool in screening for compounds that exhibit anti-viral awtvity. 5 Table 19a. Comparison of Rank order of efficacy of 12 substituted acyl guanidines in the Vpu bacterial assay and antl-fflV assay. Compound Bacterial assay p24 dIA 2 _________________________ rank order rank order (3-bromocinnamoylngganidine 110 3-(trifuoromethyl)cinnmoylguanjdine 2 2 0 3 -methZLcnno yl gu anldi ne 3 3 0 cinnamoylguanidine 4 4 0 trans -3-(I-raplh Iy)acryloylgoamdiie 5.5 7 2.25 6-methoxy-2-nahthoylI anidine 5.5 5 0.25 4-phqnlbenzoylguadine 7 11 16 (5-phenyl-penta-2,4-dienoyl)gaanidi 8 9 1 N-(3-phcnylpropanoyI)-N'- 9 .12 9 Ilexamethylene amiloride 10 6 16 2-(2-napthyl)acetoylguanidine 11 10 1 (trans-2- 12 8 1 phenylcyclopropanecarbonyl)guanidine ______ SUM diA2 61.5 Spewrian correlation coefficient 0.785 P value >0.01 Table 19b. Compounds VPu. Bacterial Atleast Ix log Ordered by p 2 4 raik order Bacterial assay rank reduction seen In score order p24 assay? (3-bromocinm~oy uanidine 4.3 1ye 3-triLBE2ormthyl jnumpoylguanidine 3.7 2ye 3-yiqcinoylZandine 3.4 3 yes einnmoy1guanidine 3.0 4 Yes trans -3-(1-naphylaeyo lgmaidine 2.9 5.5 Yes 6-metboxy-2-naghthoylguanidine 2.9 5.5 __________ 4-phenylbenzoylvundine 2.8 7 no (5pej-penta-2,4-dieno 1 g~dine 2.9 no N-(3-phenypropanoyL)-N'- 229n phenylguaidine__________________ Ilxmthylene amilod 1.9 10 no 2-2-napthyl)aeetoylguanidine 1.2 110n WO 2004/112687 PCT/AU2004/000866 -114 (tas--0.4 12 no. ph nl ylpropaecarbonyl)guanidine 0.|n 5 Example 58.4. Correlation Between Percent inhibition of MHV plaque formation and MHV-E bacterial bio-assay score. A positive correlation was seen between the activity scores assigned to compounds when tested in the Mouse Hepatitis Virus E-protein bacterial bio-assay 10 and the percent inhibition exhibited by these compounds in the Mouse Hepatitis Virus plaque assay. Example 58.5. Method: MHV plaque reduction activity data for 96 compounds screened were sorted from greatest to least percent plaque reduction and rank orders were assigned to the list of 15 compounds. This was performed for the data generated by exposure to both 10pM and 1 pM concentrations of the compounds, giving rise to two rank order lists. Similarly, a rank order list was generated for the MEIVE bacterial bioassay scores for the same 96 compounds. Where one or more compounds had the same score, the rank values for that group were averaged. 20 Spearman's statistical test for [as described in "Mathematical Statistics with Applications" (2"n edn): Mendenhall, W., Scheaffer, RL.,& Wackerly, DD. Duxbury Press, Boston Massachusetts - 1981] was used to compare rank orders. Briefly, this involved calculating the Sum of squares (SS) of the differences between rank values for each compound, and then generating the Spearman's Rank Correlation coefficient (Rs) 25 according to the formula; Rs -1-(6.SS/n(nz-1)), where n is the number of compounds ranked (96 in this case). Rs is then compared to a Table of critical values to determine statistical significance (P values). Example 58.6. Summary of Results: 30 This table summarises the Rs and P values generated as a result ofthe indicated pairwise comparisons between rank orders.

WO 2004/112687 PCT/AU2004/000866 -115 Table 20 Rs +jve correlatlor Comparison P Plague at 10!M Plaque at 1pM 0.689 >99.5 Yes Bacterial Plaque at 10gM 0,444 >99 Yes Plaque at 1pM 0.406 >98.5 Yes I Randomised order . -0..382 n/s No Example 58.7. Conclusions: The rank order comparison of 96 compounds assayed in the bacterial bio 5 assay and the antiviral assay show that MHVE bacterial assay rank order for the compounds tested is significantly positively correlated with the rank orders generated by the MHV plaque reduction assay. The significant correlation between the assays is highly indicative that either assay may be utilised to identify compounds that may be useful. The bacterial assay may thereby be a useful tool in screening for 10 compounds that exhibit anti-viral activity. Example 58.8. Correlation Between Percent Inhibition of 229E plaque formation and 229E-E bacterial bio-assay score. A positive correlation was seen between the activity scores assigned to 15 compounds when tested in the Human Coronavirus 229E f-protein bacterial bio assay and the percent inhibition exhibited by these compounds in the Human Coronavirus 229B plaque assay. Example 58.9. Method: 20 229E plaque reduction activity data for 97 compounds screened against 2.5 pM compound concentration were sorted from greatest to least percent plaque reduction and rank orders were assigned to the list of compounds. Similarly, a rank order list was generated for the 229E E bacterial bioassay scores for the same 97 compounds. Where one or more compounds had the same score, the rank values for that group were averaged. 25 Spearman's statistical test for [as described in "Mathematical Statistics with Applications"

(

2 n" edn): Mendenhall, W., Scheaffer, RL.,& Wackerly, DD. Duxbury Press, Boston WO 2004/112687 PCT/AU2004/000866 -116 Massachusetts - 1981] was used to compare rank orders. Briefly, this involves calculating the Sum of squares (SS) of the differences between rank values for each compound, and then generating the Spearman's Rank Correlation coefficient (Rs) according to the formula: Rs = 16.SS/u(nI)), where n is the number of compounds ranked (97 in this 5 case). Rs is then compared to a Table of critical values to determine statistical significance (P values). Example 58.9.1. Summary of Results and Conclusions 10 This table summarises the Rs and P values generated as a result of the indicated pairwiso comparisons between rank orders. Table 21 Rs P +ive correlatlo Comparison Plaque at 2.5pM Bacterial 0.584 >99.5 Yes randomized -0.382 n/s No The results above indicate that the 229E bacterial assay rank order for the 15 compounds tested is significantly positively correlated with the rank orders generated by the 229B plaque reduction assay. This result combined with that shown in Examples 49.1 and 49.4, provides strong evidence that either assay may be utilised to identify compounds that may be usefW. The bacterial assay may thereby be a useful tool in screening for compounds that exhibit anti-viral activity. 20 Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications The invention also includes all of the steps, features, compositions and compounds 25 referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

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Claims (41)

1. An antiviral compound of Formula I O N',R3 RI N 'kN',R2 H I or a pharmaceutically acceptable salt thereof, wherein, Ri = Re R ORd orN; R2, R3 and 14 are independently hydrogen, -121 R. o o 0 0 0 (E) N-. B N B N F F or F F and wherein X = hydrogen, hydroxy, nitro, Ci- 6 alkyl, C 3 . 4 cycloalkyl, phenyl, Ci- 6 alkeneyl, C3_ 6 cycloalkeneyl, CIr6alkeneoxy, or benzo; Rd, R 0 , Rr, Rh, Rk , RL , Rm, R., R 0 , R, independently = hydrogen, amino, C1-salkyl, hydroxy, aryl, substituted aryl, substituted amino, mono or dialkyl-substituted amino, cycloalkyl-substituted amino, aryl substituted amino, H 3 C, N4' H2N N _ NH 2 0 or PrS; Rg , Ri independently = hydrogen, hydroxy or C 1 _ 5 alkyl; R; = hydrogen, amino, Ci- 5 alkyl, hydroxy, aryl, substituted aryl, substituted amino, alkyl-substituted amino, cycloalkyl-substituted amino, aryl substituted amino, PrS, H 2 N N \ N O> ,o NH 2 0 ,or -122 when R is C 6 H 5 CH=CH, R2 is hydrogen and R3 is phenyl, R4 cannot be phenyl; when R is phenyl, R 2 is hydrogen, and R3 is benzoyl, R 4 cannot be benzoyl; when R 1 is phenyl, R 2 is substituted benzyl, R3 is hydrogen and R4 is hydrogen, Rn, R and Rp cannot all be hydrogen; when Ri is phenyl, R 3 is hydrogen and R 4 is hydrogen, R 2 cannot be benzyl or phenyl; when R is phenyl, R 2 is hydrogen, R3 cannot be phenyl together with R4 as benzoyl; when Ri is phenyl, R 2 is hydrogen, R3 and R4 cannot both be benzyl; wherein R 2 , R3, R 4 , Rj and Rk cannot all be hydrogen; and wherein the compound is not: o NH 0 NH N NH2 N NH 2 Me O NH 0 NH N NH 2 N NH 2 H H OMe OH O NH 0 NH N 0NH 2 Me N NH 2 H H NM 2 O NH 0 NH HO N JNH 2 0O 2 NN JNH H IH O NH 0 NH MeC 'I N NH- 2 N NH 2 IH 0 2 N -123 o NH 0 NH N NH 2 N NH 2 MeH H O NH 0 NH N NH 2 Me2NN NI2 H H F 3 CI or Me 2 N

2. A pharmaceutical composition comprising an antiviral compound according to claim 1 and optionally one or more pharmaceutical acceptable carriers or derivatives.

3. The pharmaceutical composition according to claim 3, further comprising one or more known antiviral compounds or molecules.

4. A method for reducing, retarding or otherwise inhibiting growth and/or replication of a virus comprising contacting a cell infected with said virus or exposed to said virus with a compound according to claim 1 or a phamaceutical composition according to claim 2 or claim 3.

5. A method for preventing the infection of a cell exposed to a virus comprising contacting said cell with a compound according to claim 1 or a pharmaceutical composition according to claim 2 or claim 3.

6. A method for the therapeutic or prophylactic treatment of a subject infected with or exposed to a virus, comprising the administration of a compound according to claim 1 or a pharmaceutical composition according to claim 2 or claim 3 to a subject in need of said treatment.

7. A method of down regulating a membrane ion channel functional activity in a cell infected with a virus, comprising contacting said cell with a compound -124 according to claim I or a pharmaceutical composition according to claim 2 or claim 3.

8. A method of reducing, retarding or otherwise inhibiting growth and/or replication of a virus that has infected a cell, said method comprising contacting said infected cell with a compound according to claim 1 or a pharmaceutical composition according to claim 2 or claim 3, wherein said compound or composition down regulates functional activity of a membrane ion channel derived from said virus and expressed in said infected cell.

9. A method of reducing, retarding or otherwise inhibiting growth and/or replication of a virus that has infected a cell in a mammal, said method comprising administering to said mammal a compound according to claim 1 or a pharmaceutical composition according to claim 2 or claim 3, wherein said compound or composition down regulates functional activity of a membrane ion channel expressed in said infected cell.

10. A method for the therapeutic or prophylactic treatment of a subject infected with or exposed to a virus comprising administering to said subject a compound according to claim 1 or a pharmaceutical composition according to claim 2 or claim 3, wherein said compound or composition down-regulates functional activity of a membrane ion channel derived from said virus.

11. The method according to any one of claims 4 to 10, wherein said virus is a Lentivirus.

12. The method according to claim 11, wherein said Lentivirus is Human Immunodeficiency Virus (HIV).

13. The method according to claim 12, wherein said compound is selected from the group consisting of: trans-3 -(1 -napthyl)acryloylguanidine, 2-ethylcinnamoylguanidine, -125 2,5-dimethyleinnamoylguanidine, 3-isopropylcinnamoylguanidine hydrochloride, (5-Phenyl-penta-2,4-dienoyl)guanidine, 3-phenylcinnamoylguanidine, 3-(cyclohex-1-en-1-yl)cinnamoylguanidine, N,N'-bis(3phenylpropanoyl)-N"-phenylguanidine, N-(3-phenylpropanoyl)-N'-phenylguanidine, 2-t-butylcinnamoylguanidine, 2-phenyleinnamoylguanidine, N-(6-Hydroxy-2-napthoyl)-N'-phenylguanidine, 3-t-butyleinnamoylguanidine, 3, 4 -(methylenedioxy)cinnamoylguanidine, 3-(2-napthyl)acryloylguanidine, 2-(1 -napthyl)acetoylguanidine, 4 -isopropylcinnamoylguanidine, N-(cinnamoyl)-N'phenylguanidine, 2- (cyclo hex-I-en-I yl)cinnamoylguanidine, 2-(2-napthyl)acetoylguanidine, 4-phenylcinnamoylguanidine, N,N'-bis-(cinnamoyl)-N"-phenylguanidine, Benzyoylguanidine, 2-cyclohexylcinnamoylguanidine, (a-Methylcinnamoyl)guanidine, cinnamoylguanidine hydrochloride, 1-napthoylguanidine, (3-phenylpropanoyl)guanidine, 4-t-butylcinnamoylguanidine, N,N'-Bis(3-phenylpropanoyl)guanidine and N-Benzoyl-N'-cinnamoylguanidine.

14. The method according to claim 12, wherein said compound is (5-Phenyl penta-2,4-dienoyl)guanidine. -126

15. The method according to any one of claims 12 to 14, wherein said HIV is HIV-1.

16. The method according to any one of claims 4 to 10 wherein said virus is a Coronavirus.

17. The method according to claim 16, wherein said Coronavirus is the Severe Acute Respiratory Syndrome virus (SARS).

18. The method according to claim 17, wherein said compound is selected from the group consisting of 4 -t-butylcinnamoylguanidine, 3-(2-napthyl)acryloylguanidine, 3-(cyclohex- 1-en-I -yl)cinnamoylguanidine, 2,5-dimethylcinnamoylguanidine, trans-3-(I -napthyl)acryloylguanidine, 4-isopropyleinnamoylguanidine, 3-phenylcinnamoylguanidine, 4-phenylcinnamoylguanidine, 3-t-butylcinnamoylguanidine, 2 -t-butylcinnamoylguanidine, 2 -cyclohexylcinnamoylguanidine, N-(3-phenylpropanoyl)-N'-phenylguanidine, 2-(1 -napthyl)acetoylguanidine, 2-ethylcinnamoylguanidine, N-( 6 -Hydroxy-2-napthoyl)-N'-phenylguanidine, N,N'-bis(3phenylpropanoyl)-N"-phenylguanidine, I -napthoylguanidine, 3, 4 -(methylenedioxy)cinnamoylguanidine, N-(cinnamoyl)-N'phenylguanidine, (3-phenylpropanoyl)guanidine, 2 -(2-napthyl)acetoylguanidine, (a-Methylcinnamoyl)guanidine, -127 (5-Phenyl-penta-2,4-dienoyl)guanidine, (Quinoline-2-carbonyl)guanidine, N,N'-Bis(amidino)napthalene-2,6-dicarboxamide, N-Benzoyl-N'-cinnamoylguanidine and N-(2-napthoyl)-N'-phenylguanidine.

19. The method according to claim 17, wherein said compound is trans-3-(1 napthyl)acryloylguanidine.

20. The method according to claim 16, wherein said Coronavirus is human Coronavirus 229E.

21. The method according to claim 20, wherein said compound is selected from the group consisting of 4-isopropylcinnamoylguanidine, 4-t-butylcinnamoylguanidine, 3-isopropylcinnamoylguanidine hydrochloride, 3-t-butyleinnamoylguanidine, 2-t-butylcinnamoylguanidine, trans-3-(1-napthyl)acryloylguanidine, 3-(2-napthyl)acryloylguanidine, 2-phenyleinnamoylguanidine, 3-phenyleinnamoylguanidine, 3-(cyclohex-l-en-1-yl)cinnamoylguanidine, 2-(cyclohex-1-en-lyl)cinnamoylguanidine, 1-napthoylguanidine, (5-Phenyl-penta-2,4-dienoyl)guanidine, 2,5-dimethylcinnamoylguanidine, (a-Methylcinnamoyl)guanidine, 4-phenylcinnamoylguanidine, 2-(1 -napthyl)acetoylguanidine, 2-ethylcinnamoylguanidine, 2-cyclohexylcinnamoylguanidine, -128 N,N'-Bis(amidino)napthalene-2,6-dicarboxamide, N,N'-Bis(3-phenylpropanoyl)guanidine, N,N'-bis(3phenylpropanoyl)-N"-phenylguanidine, 2-(2-napthyl)acetoylguanidine, N-(3-phenylpropanoyl)-N'-phenylguanidine, N-Benzoyl-N'-cinnamoylguanidine, N-(cinnamoyl)-N'phenylguanidine, Cinnamoylguanidine, (3-phenylpropanoyl)guanidine, Benzyoylguanidine, (Quinoline-2-carbonyl)guanidine, N-(2-napthoyl)-N'-phenylguanidine and

22. The method according to claim 20, wherein said compound is selected from the group consisting of 2-t-butylcinnamoylguanidine, 4 -isopropylcinnamoylguanidine, 2-(cyclohex- 1-en-i yl)cinnamoylguanidine, 2-cyclohexylcinnamoylguanidine, 2-phenylcinnamoylguanidine, 4-t-butylcinnamoylguanidine, 3-phenylcinnamoylguanidine, trans-3-(1-napthyl)acryloylguanidine, 3 -(2-napthyl)acryloylguanidine, 3 -(cyclohex- 1-en-1 -yl)cinnamoylguanidine, (a-Methylcinnamoyl)guanidine, 2,5-dimethylcinnamoylguanidine, 3-t-butylcinnamoylguanidine, 3-isopropylcinnamoylguanidine hydrochloride, (5-Phenyl-penta-2,4-dienoyl)guanidine, N-(3-phenylpropanoyl)-N'-Phenylguanidine, 3, 4 -(methylenedioxy)cinnamoylguanidine, N,N'-Bis(amidino)napthalene-2,6-Dicarboxamide, -129 N,N'-Bis(3-phenylpropanoyl)guanidine, 1-napthoylguanidine, 2-(1 -napthyl)acetoylguanidine, 2-(2-napthyl)acetoylguanidine and N,N'-bis(3phenylpropanoyl)-N"-Phenylguanidine.

23. The method according to claim 16, wherein said Coronavirus is human Coronavirus OC43.

24. The method according to claim 23, wherein said compound is selected from the group consisting of trans-3-(1 -napthyl)acryloylguanidine, 4-isopropylcinnamoylguanidine, (5-Phenyl-penta-2,4-dienoyl)guanidine and 2-t-butylcinnamoylguanidine.

25. The method according to claim 16, wherein said Coronavirus is porcine respiratory Coronavirus (PRCV).

26. The method according to claim 25, wherein said compound is selected from the group consisting of N-(3-phenylpropanoyl)-N'-phenylguanidine, trans-3 -(1 -napthyl)acryloylguanidine and 2-(2-napthyl)acetoylguanidine.

27. The method according to claim 16, wherein said Coronavirus is bovine Coronvirus (BCV).

28. The method according to claim 27, wherein said compound is selected from the group consisting of trans-3-(1-napthyl)acryloylguanidine, (5-Phenyl-penta-2,4-dienoyl)guanidine, 2-(2-napthyl)acetoylguanidine and -130 N-(3-phenylpropanoyl)-N'-phenylguanidine.

29. The method according to claim 16, wherein said Coronavirus is any one of the known coronavirus isolates listed in Table 1.

30. The method according to claim 29, wherein said compound is selected f-rom the group consisting of 4 -isopropyleinnamoylguanidine, 4 -t-butylcinnamoylguanidine and 3-isopropyleinnamoylguanidine hydrochloride.

31. The method according to any one of claims 4 to 10, wherein said virus is the Hepatitis C virus.

32. The method according to claim 31, wherein said compound is selected from the group consisting of 2,5-dimethylcinnamoylguanidine, 4-isopropyleinnamoylguanidine, trans-3-(1 -napthyl)acryloylguanidine, 4-t-butylcinnamoylguanidine, 2-t-butylcinnamoylguanidine, 2-ethylcinnamoylguanidine, 2-cyclohexyleinnamoylguanidine, 1-napthoylguanidine, 3-t-butylcinnamoylguanidine, (5-Phenyl-penta-2,4-dienoyl)guanidine, N-(cinnamoyl)-N'phenylguanidine, 3-isopropyleinnamoylguanidine hydrochloride, N-(3-phenylpropanoyl)-N'-phenylguanidine, N,N'-bis(3phenylpropanoyl)-N"-phenylguanidine, 3-(2-napthyl)acryloylguanidine, 4 -phenyleinnamoylguanidine, N-(6-Hydroxy-2-napthoyl)-N'-phenylguanidine, -131 3-(cyclohex-1 -en-1-yl)cinnamoylguanidine, 2-(1 -napthyl)acetoylguanidine, (a-Methylcinnamoyl)guanidine, 3,4-(methylenedioxy)cinnamoylguanidine, 2-phenylcinnamoylguanidine, 2-(cyclohex- 1-en-i yl)cinnamoylguanidine, 3-phenylcinnamoylguanidine, (3-phenylpropanoyl)guanidine and (4-Nitrocinnamoyl)guanidine.

33. The method according to any one of claims 4 to 10, wherein said virus is Equine Arteritis virus.

34. The method according to claim 33, wherein said compound is selected from the group consisting of trans-3-(1-napthyl)acryloylguanidine, 2-t-butyleinnamoylguanidine and 2-(cyclohex- 1-en-I yl)cinnamoylguanidine.

35. An antiviral compound selected from the group consisting of N,N'-bis(1-napthoyl)guanidine, N-(cinnamoyl)-N'phenylguanidine, (3-phenylpropanoyl)guanidine, N,N'-bis-(cinnamoyl)-N"-phenylguanidine, N-(3-phenylpropanoyl)-N'-phenylguanidine, N,N'-bis(3phenylpropanoyl)-N"-phenylguanidine, N-(6-Hydroxy-2-napthoyl)-N'-phenylguanidine, N,N'-Bis(amidino)napthalene-2,6-dicarboxamide, N"-Cinnamoyl-N,N'-diphenylguanidine, N,N'-Bis(3-phenylpropanoyl)guanidine, benzyoylguanidine, N-benzoyl-N'-cinnamoylguanidine, (5-Phenyl-penta-2,4-dienoyl)guanidine and -132 (Quinoline-2-carbonyl)guanidine, or a pharmaceutically acceptable salt thereof.

36. A pharmaceutical composition comprising a compound according to claim 35, and optionally one or more pharmaceutical acceptable carriers or derivatives.

37. The pharmaceutical composition according to claim 36, further comprising one or more known antiviral compounds.

38. The method according to any one of claims 4 to 10, wherein said compound is selected from the antiviral compounds according to claim 35.

39. The method according to any one of claims 4 to 10, wherein said virus is Dengue virus and said compound is trans -3-(-napthyl)acryloylguanidine.

40. Use of a compound according to claim 1 or claim 35 for the manufacture of a medicament for: reducing, retarding or otherwise inhibiting growth and/or replication of a virus; preventing the infection of a cell exposed to a virus; the therapeutic or prophylactic treatment of a subject infected with or exposed to a virus; down regulating a membrane ion channel functional activity in a cell infected with a virus; reducing, retarding or otherwise inhibiting growth and/or replication of a virus that has infected a cell; reducing, retarding or otherwise inhibiting growth and/or replication of a virus that has infected a cell in a mammal; or the therapeutic or prophylactic treatment of a subject infected with or exposed to a virus. -133

41. A compound according to claim 1 or claim 35; a method according to any one of claims 4 to 10; or use according to claim 40, substantially as herein described with reference to any one or more of the examples but excluding comparative examples.